Variant nucleic acid libraries for GLP1 receptor

ABSTRACT

Provided herein are methods and compositions relating to glucagon-like peptide-1 receptor (GLP1R) libraries having nucleic acids encoding for a scaffold comprising a GLP1R binding domain. Libraries described herein include variegated libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries generated when the nucleic acid libraries are translated. Further described herein are cell libraries expressing variegated nucleic acid libraries described herein.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Patent Application No. 62/810,377 filed on Feb. 26, 2019; U.S. Provisional Patent Application No. 62/830,316 filed on Apr. 5, 2019; U.S. Provisional Patent Application No. 62/855,836 filed on May 31, 2019; U.S. Provisional Patent Application No. 62/904,563 filed on Sep. 23, 2019; U.S. Provisional Patent Application No. 62/945,049 filed on Dec. 6, 2019; and U.S. Provisional Patent Application No. 62/961,104 filed on Jan. 14, 2020, each of which is incorporated by reference in its entirety.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 31, 2020, is named 44854-787_201_SL.txt and is 1,080,872 bytes in size.

BACKGROUND

G protein-coupled receptors (GPCRs) are implicated in a wide variety of diseases. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. Thus, there is a need for improved agents for therapeutic intervention which target GPCRs.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are antibodies or antibody fragments thereof that binds GLP1R, comprising an immunoglobulin heavy chain and an immunoglobulin light chain: (a) wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321; and (b) wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2303; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2310. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2304; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2311. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2305; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2312. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2306; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2313. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2307; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2314. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2308; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2315. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2309, 2317, 2318, 2319; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2316. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)₂ fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody or antibody fragment thereof is chimeric or humanized. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody has an EC50 less than about 25 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody has an EC50 less than about 20 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody has an EC50 less than about 10 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody is an agonist of GLP1R. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody is an antagonist of GLP1R. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody is an allosteric modulator of GLP1R. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the allosteric modulator of GLP1R is a negative allosteric modulator. Further provided herein are antibodies or antibody fragments thereof that binds GLP1R, wherein the antibody or antibody fragment comprises a CDR-H3 comprising a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, or 2302.

Provided herein are nucleic acid libraries comprising a plurality of nucleic acids, wherein each nucleic acid encodes for a sequence that when translated encodes for an immunoglobulin scaffold, wherein the immunoglobulin scaffold comprises a CDR-H3 loop that comprises a GLP1R binding domain, and wherein each nucleic acid comprises a sequence encoding for a sequence variant of the GLP1R binding domain. Further provided herein are nucleic acid libraries, wherein a length of the CDR-H3 loop is about 20 to about 80 amino acids. Further provided herein are nucleic acid libraries, wherein a length of the CDR-H3 loop is about 80 to about 230 base pairs. Further provided herein are nucleic acid libraries, wherein the immunoglobulin scaffold further comprises one or more domains selected from variable domain, light chain (VL), variable domain, heavy chain (VH), constant domain, light chain (CL), and constant domain, heavy chain (CH). Further provided herein are nucleic acid libraries, wherein the VH domain is IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, or IGHV4-59/61. Further provided herein are nucleic acid libraries, wherein the VH domain is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. Further provided herein are nucleic acid libraries, wherein the VH domain is IGHV1-69 and IGHV3-30. Further provided herein are nucleic acid libraries, wherein the VL domain is IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, or IGLV3-1. Further provided herein are nucleic acid libraries, wherein a length of the VH domain is about 90 to about 100 amino acids. Further provided herein are nucleic acid libraries, wherein a length of the VL domain is about 90 to about 120 amino acids. Further provided herein are nucleic acid libraries, wherein a length of the VH domain is about 280 to about 300 base pairs. Further provided herein are nucleic acid libraries, wherein a length of the VL domain is about 300 to about 350 base pairs. Further provided herein are nucleic acid libraries, wherein the library comprises at least 10⁵ non-identical nucleic acids. Further provided herein are nucleic acid libraries, wherein the immunoglobulin scaffold comprises a single immunoglobulin domain. Further provided herein are nucleic acid libraries, wherein the immunoglobulin scaffold comprises a peptide of at most 100 amino acids.

Provided herein are protein libraries comprising a plurality of proteins, wherein each of the proteins of the plurality of proteins comprise an immunoglobulin scaffold, wherein the immunoglobulin scaffold comprises a CDR-H3 loop that comprises a sequence variant of a GLP1R binding domain. Further provided herein are protein libraries, wherein a length of the CDR-H3 loop is about 20 to about 80 amino acids. Further provided herein are protein libraries, wherein the immunoglobulin scaffold further comprises one or more domains selected from variable domain, light chain (VL), variable domain, heavy chain (VH), constant domain, light chain (CL), and constant domain, heavy chain (CH). Further provided herein are protein libraries, wherein the VH domain is IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, or IGHV4-59/61. Further provided herein are protein libraries, wherein the VH domain is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. Further provided herein are protein libraries, wherein the VH domain is IGHV1-69 and IGHV3-30. Further provided herein are protein libraries, wherein the VL domain is IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, or IGLV3-1. Further provided herein are protein libraries, wherein a length of the VH domain is about 90 to about 100 amino acids. Further provided herein are protein libraries, wherein a length of the VL domain is about 90 to about 120 amino acids. Further provided herein are protein libraries, wherein the plurality of proteins are used to generate a peptidomimetic library. Further provided herein are protein libraries, wherein the protein library comprises antibodies.

Provided herein are protein libraries comprising a plurality of proteins, wherein the plurality of proteins comprises sequence encoding for different GPCR binding domains, and wherein the length of each GPCR binding domain is about 20 to about 80 amino acids. Further provided herein are protein libraries, wherein the protein library comprises peptides. Further provided herein are protein libraries, wherein the protein library comprises immunoglobulins. Further provided herein are protein libraries, wherein the protein library comprises antibodies. Further provided herein are protein libraries, wherein the plurality of proteins is used to generate a peptidomimetic library.

Provided herein are vector libraries comprising a nucleic acid library as described herein.

Provided herein are cell libraries comprising a nucleic acid library as described herein.

Provided herein are cell libraries comprising a protein library as described herein.

Provided herein are antibodies, wherein the antibody comprises a CDR-H3 comprising a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, or 2302.

Provided herein are antibodies, wherein the antibody comprises a CDR-H3 comprising a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, or 2302; and wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)₂ fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.

Provided herein are methods of inhibiting GLP1R activity, comprising administering an antibody or antibody fragment as described herein. Further provided herein are methods of inhibiting GLP1R activity, wherein the antibody or antibody fragment is an allosteric modulator. Further provided herein are methods of inhibiting GLP1R activity, wherein the antibody or antibody fragment is a negative allosteric modulator. Further provided herein are methods of treatment of a metabolic disorder, comprising administering to a subject in need thereof an antibody or antibody fragment as described herein. Further provided herein are methods of treatment of a metabolic disorder, wherein the metabolic disorder is Type II diabetes or obesity.

Provided herein are nucleic acid libraries, comprising: a plurality of nucleic acids, wherein each of the nucleic acids encodes for a sequence that when translated encodes for a GLP1R binding immunoglobulin, wherein the GLP1R binding immunoglobulin comprises a variant of a GLP1R binding domain, wherein the GLP1R binding domain is a ligand for the GLP1R, and wherein the nucleic acid library comprises at least 10,000 variant immunoglobulin heavy chains and at least 10,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 50,000 variant immunoglobulin heavy chains and at least 50,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 100,000 variant immunoglobulin heavy chains and at least 100,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 10⁵ non-identical nucleic acids. Further provided herein are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when translated is about 90 to about 100 amino acids. Further provided herein are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when translated is about 100 to about 400 amino acids. Further provided herein are nucleic acid libraries, wherein the variant immunoglobulin heavy chain when translated comprises at least 80% sequence identity to SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321. Further provided herein are nucleic acid libraries, wherein the variant immunoglobulin light chain when translated comprises at least 80% sequence identity to SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316.

Provided herein are nucleic acid libraries comprising: a plurality of nucleic acids, wherein each of the nucleic acids encodes for a sequence that when translated encodes for a GLP1R single domain antibody, wherein each sequence of the plurality of sequences comprises a variant sequence encoding for at least one of a CDR1, CDR2, and CDR3 on a heavy chain; wherein the library comprises at least 30,000 variant sequences; and wherein the antibody or antibody fragments bind to its antigen with a K_(D) of less than 100 nM. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 50,000 variant immunoglobulin heavy chains and at least 50,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 100,000 variant immunoglobulin heavy chains and at least 100,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 10⁵ non-identical nucleic acids. Further provided herein are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when translated is about 90 to about 100 amino acids. Further provided herein are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when translated is about 100 to about 400 amino acids. Further provided herein are nucleic acid libraries, wherein the variant immunoglobulin heavy chain when translated comprises at least 80% sequence identity to SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321. Further provided herein are nucleic acid libraries, wherein the variant immunoglobulin light chain when translated comprises at least 80% sequence identity to SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316.

Provided herein antagonists of GLP1R comprising SEQ ID NO: 2279 or 2320. Further provided herein are antagonists, wherein the antagonist comprises an EC50 of no more than 1.5 nM. Further provided herein are antagonists, wherein the antagonist comprises an EC50 of no more than 1.0 nM. Further provided herein are antagonists, wherein the antagonist comprises an EC50 of no more than 0.5 nM. Further provided herein are antagonists, wherein the antagonist is an antibody or antibody fragment thereof.

Provided herein are nucleic acid libraries, comprising: a plurality of nucleic acids, wherein each of the nucleic acids encodes for a sequence that when translated encodes for a GLP1R binding immunoglobulin, wherein the GLP1R binding immunoglobulin comprises a variant of a GLP1R binding domain, wherein the GLP1R binding domain is a ligand for the GLP1R, and wherein the nucleic acid library comprises at least 10,000 variant immunoglobulin heavy chains and at least 10,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 50,000 variant immunoglobulin heavy chains and at least 50,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 100,000 variant immunoglobulin heavy chains and at least 100,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 10⁵ non-identical nucleic acids. Further provided herein are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when translated is about 90 to about 100 amino acids. Further provided herein are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when translated is about 100 to about 400 amino acids. Further provided herein are nucleic acid libraries, wherein the variant immunoglobulin heavy chain when translated comprises at least 90% sequence identity to SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321. Further provided herein are nucleic acid libraries, wherein the variant immunoglobulin light chain when translated comprises at least 90% sequence identity to SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316.

Provided herein are nucleic acid libraries comprising: a plurality of nucleic acids, wherein each of the nucleic acids encodes for a sequence that when translated encodes for a GLP1R single domain antibody, wherein each sequence of the plurality of sequences comprises a variant sequence encoding for at least one of a CDR1, CDR2, and CDR3 on a heavy chain; wherein the library comprises at least 30,000 variant sequences; and wherein the antibody or antibody fragments bind to its antigen with a K_(D) of less than 100 nM. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 50,000 variant immunoglobulin heavy chains and at least 50,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 100,000 variant immunoglobulin heavy chains and at least 100,000 variant immunoglobulin light chains. Further provided herein are nucleic acid libraries, wherein the nucleic acid library comprises at least 10⁵ non-identical nucleic acids. Further provided herein are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when translated is about 90 to about 100 amino acids. Further provided herein are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when translated is about 100 to about 400 amino acids. Further provided herein are nucleic acid libraries, wherein the variant immunoglobulin heavy chain when translated comprises at least 90% sequence identity to SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321. Further provided herein are nucleic acid libraries, wherein the variant immunoglobulin light chain when translated comprises at least 90% sequence identity to SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316.

Provided herein are antibodies or antibody fragments that binds GLP1R, comprising an immunoglobulin heavy chain and an immunoglobulin light chain: (a) wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321; and (b) wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316. Further provided herein are antibodies or antibody fragments, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2303; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2310. Further provided herein are antibodies or antibody fragments, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2304; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2311. Further provided herein are antibodies or antibody fragments, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2305; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2312. Further provided herein are antibodies or antibody fragments, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2306; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2313. Further provided herein are antibodies or antibody fragments, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2307; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2314. Further provided herein are antibodies or antibody fragments, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2308; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2315. Further provided herein are antibodies or antibody fragments, wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2309; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in SEQ ID NO: 2316. Further provided herein are antibodies or antibody fragments, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment thereof is chimeric or humanized. Further provided herein are antibodies or antibody fragments, wherein the antibody has an EC50 less than about 25 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody has an EC50 less than about 20 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody has an EC50 less than about 10 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody is an agonist of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the antibody is an antagonist of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the antibody is an allosteric modulator of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the allosteric modulator of GLP1R is a negative allosteric modulator.

Provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment comprises a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, or 2302 or a sequence set forth in Table 27.

Provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment comprises a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, or 2302 or a sequence set forth in Table 27; and wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)₂ fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof.

Provided herein are antagonists of GLP1R comprising SEQ ID NO: 2279 or 2320. Further provided herein are antagonists of GLP1R, wherein the antagonist comprises an EC50 of no more than 1.5 nM. Further provided herein are antagonists of GLP1R, wherein the antagonist comprises an EC50 of no more than 1.0 nM. Further provided herein are antagonists of GLP1R, wherein the antagonist comprises an EC50 of no more than 0.5 nM. Further provided herein are antagonists of GLP1R, wherein the antagonist is an antibody or antibody fragment.

Provided herein are agonists of GLP1R comprising SEQ ID NO: 2317. Further provided herein are agonists of GLP1R, wherein the agonist comprises an EC50 of no more than 1.5 nM. Further provided herein are agonists of GLP1R, wherein the agonist comprises an EC50 of no more than 1.0 nM. Further provided herein are agonists of GLP1R, wherein the agonist comprises an EC50 of no more than 0.5 nM. Further provided herein are agonists of GLP1R, wherein the agonist is an antibody or antibody fragment.

Provided herein are methods of inhibiting GLP1R activity, comprising administering the antibody or antibody fragment as described herein. Further provided herein are methods of inhibiting GLP1R activity, wherein the antibody or antibody fragment is an allosteric modulator. Further provided herein are methods of inhibiting GLP1R activity, wherein the antibody or antibody fragment is a negative allosteric modulator.

Provided herein are methods for treatment of a metabolic disorder, comprising administering to a subject in need thereof the antibody as described herein. Provided herein are methods for treatment of a metabolic disorder, wherein the metabolic disorder is Type II diabetes or obesity.

Provided herein are protein libraries encoded by the nucleic acid library as described herein, wherein the protein library comprises peptides. Further provided herein are protein libraries, wherein the protein library comprises immunoglobulins. Further provided herein are protein libraries, wherein the protein library comprises antibodies. Further provided herein are protein libraries, wherein the protein library is a peptidomimetic library.

Provided herein are vector libraries comprising the nucleic acid library as described herein. Provided herein are cell libraries comprising the nucleic acid library as described herein. Provided herein are cell libraries comprising the protein library as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a first schematic of an immunoglobulin scaffold.

FIG. 1B depicts a second schematic of an immunoglobulin scaffold.

FIG. 2 depicts a schematic of a motif for placement in a scaffold.

FIG. 3 presents a diagram of steps demonstrating an exemplary process workflow for gene synthesis as disclosed herein.

FIG. 4 illustrates an example of a computer system.

FIG. 5 is a block diagram illustrating an architecture of a computer system.

FIG. 6 is a diagram demonstrating a network configured to incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS).

FIG. 7 is a block diagram of a multiprocessor computer system using a shared virtual address memory space.

FIG. 8A depicts a schematic of an immunoglobulin scaffold comprising a VH domain attached to a VL domain using a linker.

FIG. 8B depicts a schematic of a full-domain architecture of an immunoglobulin scaffold comprising a VH domain attached to a VL domain using a linker, a leader sequence, and pIII sequence.

FIG. 8C depicts a schematic of four framework elements (FW1, FW2, FW3, FW4) and the variable 3 CDR (L1, L2, L3) elements for a VL or VH domain.

FIGS. 9A-90 depict the cell binding data for GLP1R-2 (FIG. 9A), GLP1R-3 (FIG. 9B), GLP1R-8 (FIG. 9C), GLP1R-26 (FIG. 9D), GLP1R-30 (FIG. 9E), GLP1R-56 (FIG. 9F), GLP1R-58 (FIG. 9G), GLP1R-10 (FIG. 9H), GLP1R-25 (FIG. 9I), GLP1R-60 (FIG. 9J), GLP1R-70 (FIG. 9K), GLP1R-72 (FIG. 9L), GLP1R-83 (FIG. 9M), GLP1R-93 (FIG. 9N), and GLP1R-98 (FIG. 9O).

FIGS. 10A-100 depict graphs of GLP1R-2 (FIG. 10A), GLP1R-3 (FIG. 10B), GLP1R-8 (FIG. 10C), GLP1R-26 (FIG. 10D), GLP1R-30 (FIG. 10E), GLP1R-56 (FIG. 10F), GLP1R-58 (FIG. 10G), GLP1R-10 (FIG. 10H), GLP1R-25 (FIG. 10I), GLP1R-60 (FIG. 10J), GLP1R-70 (FIG. 10K), GLP1R-72 (FIG. 10L), GLP1R-83 (FIG. 10M), GLP1R-93 (FIG. 10N), and GLP1R-98 (FIG. 10O) variants on inhibition of GLP1-7-36 peptide induced cAMP activity.

FIGS. 11A-11G depict cell functional data for GLP1R-2 (FIG. 11A), GLP1R-3 (FIG. 11B), GLP1R-8 (FIG. 11C), GLP1R-26 (FIG. 11D), GLP1R-30 (FIG. 11E), GLP1R-56 (FIG. 11F), and GLP1R-58 (FIG. 11G).

FIGS. 12A-12G depict graphs of GLP1R-2 (FIG. 12A), GLP1R-3 (FIG. 12B), GLP1R-8 (FIG. 12C), GLP1R-26 (FIG. 12D), GLP1R-30 (FIG. 12E), GLP1R-56 (FIG. 12F), and GLP1R-58 (FIG. 12G) variants on inhibition of Exendin-4 peptide induced cAMP activity.

FIG. 13 depicts a schematic of glucagon (SEQ ID NO: 2740), GLP1-1 (SEQ ID NO: 6), and (GLP-2 SEQ ID NO: 2741).

FIGS. 14A-14C depict cell-binding affinity of purified immunoglobulins.

FIG. 14D depicts cAMP activity of purified immunoglobulins.

FIGS. 15A-15H depict binding curves plotting IgG concentrations in nanomolar (nM) against MFI (mean fluorescence intensity) for GLP1R-238 (FIG. 15A), GLP1R-240 (FIG. 15B), GLP1R-241 (FIG. 15C), GLP1R-242 (FIG. 15D), GLP1R-243 (FIG. 15E), GLP1R-244 (FIG. 15F), pGPCR-GLP1R-43 (FIG. 15G), and pGPCR-GLP1R-44 (FIG. 15H).

FIGS. 16A-161 depict flow cytometry data of binding assays presented as dot plots with 100 nM IgG of GLP1R-238 (FIG. 16A), GLP1R-240 (FIG. 16B), GLP1R-241 (FIG. 16C), GLP1R-242 (FIG. 16D), GLP1R-243 (FIG. 16E), GLP1R-244 (FIG. 16F), pGPCR-GLP1R-43 (FIG. 16G), pGPCR-GLP1R-44 (FIG. 16H), and GLP1R-239 (FIG. 16I).

FIGS. 17A-17B depict data from cAMP assays with relative luminescence units (RLU) on the y-axis and concentration in nanomolar (nM) on the x-axis. cAMP was measured in response to GLP1 (7-36), GLP1R-238, GLP1R-239, GLP1R-240, GLP1R-241, GLP1R-242, GLP1R-243, GLP1R-244, pGPCR-GLP1R-43, pGPCR-GLP1R-44, and buffer.

FIG. 17C depicts a graph of cAMP allosteric effect of GLP1R-241.

FIG. 17D depicts a graph of beta-arrestin recruitment of GLP1R-241.

FIG. 17E depicts a graph of GLP1R-241 internalization.

FIGS. 18A-18B depict data from cAMP assays with relative luminescence units (RLU) on the y-axis and concentration of GLP1 (7-36) in nanomolar (nM) on the x-axis. Allosteric effects of GLP1R-238, GLP1R-239, GLP1R-240, GLP1R-241, GLP1R-242, GLP1R-243, GLP1R-244, pGPCR-GLP1R-43, pGPCR-GLP1R-44, and no antibody were tested.

FIGS. 19A-19F depict flow cytometry data of binding assays presented as dot plots and histograms for GLP1R-59-2 (FIG. 19A), GLP1R-59-241 (FIG. 19B), GLP1R-59-243 (FIG. 19C), GLP1R-3 (FIG. 19D), GLP1R-241 (FIG. 19E), and GLP1R-2 (FIG. 19F). FIGS. 19A-19F also depict titration curves plotting IgG concentrations in nanomolar (nM) against MFI (mean fluorescence intensity) for GLP1R-59-2 (FIG. 19A), GLP1R-59-241 (FIG. 19B), GLP1R-59-243 (FIG. 19C), GLP1R-3 (FIG. 19D), GLP1R-241 (FIG. 19E), and GLP1R-2 (FIG. 19F).

FIGS. 20A-20F depict data from cAMP assays with relative luminescence units (RLU) on the y-axis and concentration of GLP1 (7-36) in nanomolar (nM) on the x-axis as well as beta-arrestin recruitment and receptor internalization for GLP1R-59-2 (FIG. 20A), GLP1R-59-241 (FIG. 20B), GLP1R-59-243 (FIG. 20C), GLP1R-3 (FIG. 20D), GLP1R-241 (FIG. 20E), and GLP1R-2 (FIG. 20F).

FIGS. 21A-21B depicts graphs of TIGIT affinity distribution for the VHH libraries, depicting either the affinity threshold from 20 to 4000 (FIG. 21A) or the affinity threshold from 20 to 1000 (FIG. 21B). Out of 140 VHH binders, 51 variants were <100 nM and 90 variants were <200 nM.

FIGS. 22A-22B depict graphs of FACs analysis (FIG. 22A) and graphs of a dose curve and specificity (FIG. 22B) of GLP1R-43-77.

FIG. 23A depicts a schema of heavy chain IGHV3-23 design. FIG. 23 A discloses SEQ ID NOS 2742-2747, respectively, in order of appearance.

FIG. 23B depicts a schema of heavy chain IGHV1-69 design. FIG. 23B discloses SEQ ID NOS 2748-2753, respectively, in order of appearance.

FIG. 23C depicts a schema of light chains IGKV 2-28 and IGLV 1-51 design. FIG. 23C discloses SEQ ID NOS 2754-2759, respectively, in order of appearance.

FIG. 23D depicts a schema of the theoretical diversity and final diversity of a GLP1R library.

FIGS. 23E-23F depict graphs of FACS binding of GLP1R IgGs.

FIGS. 23G-23H depict graphs of cAMP assays using purified GLP1R IgGs.

FIG. 24A depicts a graph of GLP1R-3 inhibition as compared to no antibody. Relative luminescence units (RLU) is depicted on the y-axis, and concentration of GLP1 (7-36) is depicted in nanomolar (nM) on the x-axis.

FIG. 24B depicts a graph of GLP1R-3 inhibition at high concentrations following stimulation with 0.05 nM GLP1 (7-36). Relative luminescence units (RLU) is depicted on the y-axis, and concentration of GLP1R-3 is depicted in nanomolar (nM) on the x-axis.

FIG. 24C depicts glucose levels after glucose administration when treated with vehicle (triangles), liraglutide (squares), and GLP1R-3 (circles) in a mouse model of diet induced obesity.

FIG. 24D depicts glucose levels after glucose administration when treated with vehicle (open triangles), liraglutide (squares), and GLP1R-59-2 (closed triangles) in a mouse model of diet induced obesity.

FIG. 25A depicts a graph of the blood glucose levels in mice (mg/dL; y-axis) treated with GLP1R-59-2 (agonist), GLP1R-3 (antagonist), and control over time (in minutes, x-axis).

FIG. 25B depicts a graph of blood glucose levels in mice (mg/dL; y-axis) treated with GLP1R-59-2 (agonist), GLP1R-3 (antagonist), and control.

FIG. 25C depicts a graph of the blood glucose levels (mg/dL; y-axis) in GLP1R-59-2 (agonist) treated mice in both the fasted (p=0.0008) and non-fasted (p<0.0001) mice compared to control.

FIG. 25D depicts a graph of the blood glucose levels (mg/dL/min; y-axis) in pre-dosed GLP1R-59-2 (agonist), GLP1R-3 (antagonist), and control mice.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques, which are within the skill of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

Definitions

Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/−10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

Unless specifically stated, as used herein, the term “nucleic acid” encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid sequences, when provided, are listed in the 5′ to 3′ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids. A “nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length. Moreover, provided herein are methods for the synthesis of any number of polypeptide-segments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide-synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, including non-coding DNA or RNA, such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for a gene or gene fragment referred herein may comprise at least one region encoding for exon sequences without an intervening intron sequence in the genomic equivalent sequence.

GPCR Libraries for GLP1 Receptor

Provided herein are methods and compositions relating to G protein-coupled receptor (GPCR) binding libraries for glucagon-like peptide-1 receptor (GLP1R) comprising nucleic acids encoding for a scaffold comprising a GPCR binding domain. Scaffolds as described herein can stably support a GPCR binding domain. The GPCR binding domain may be designed based on surface interactions of a GLP1R ligand and GLP1R. Libraries as described herein may be further variegated to provide for variant libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries that may be generated when the nucleic acid libraries are translated. In some instances, nucleic acid libraries as described herein are transferred into cells to generate a cell library. Also provided herein are downstream applications for the libraries synthesized using methods described herein. Downstream applications include identification of variant nucleic acids or protein sequences with enhanced biologically relevant functions, e.g., improved stability, affinity, binding, functional activity, and for the treatment or prevention of a disease state associated with GPCR signaling.

Scaffold Libraries

Provided herein are libraries comprising nucleic acids encoding for a scaffold, wherein sequences for GPCR binding domains are placed in the scaffold. Scaffold described herein allow for improved stability for a range of GPCR binding domain encoding sequences when inserted into the scaffold, as compared to an unmodified scaffold. Exemplary scaffolds include, but are not limited to, a protein, a peptide, an immunoglobulin, derivatives thereof, or combinations thereof. In some instances, the scaffold is an immunoglobulin. Scaffolds as described herein comprise improved functional activity, structural stability, expression, specificity, or a combination thereof. In some instances, scaffolds comprise long regions for supporting a GPCR binding domain.

Provided herein are libraries comprising nucleic acids encoding for a scaffold, wherein the scaffold is an immunoglobulin. In some instances, the immunoglobulin is an antibody. As used herein, the term antibody will be understood to include proteins having the characteristic two-armed, Y-shape of a typical antibody molecule as well as one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv) (including fragments in which the VL and VH are joined using recombinant methods by a synthetic or natural linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, including single chain Fab and scFab), a single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH, CL, and CH1 domains), a F(ab′)₂ fragment (including bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fd fragment (including fragments comprising the VH and CH1 fragment), a Fv fragment (including fragments comprising the VL and VH domains of a single arm of an antibody), a single-domain antibody (dAb or sdAb) (including fragments comprising a VH domain), an isolated complementarity determining region (CDR), a diabody (including fragments comprising bivalent dimers such as two VL and VH domains bound to each other and recognizing two different antigens), a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some instances, the libraries disclosed herein comprise nucleic acids encoding for a scaffold, wherein the scaffold is a Fv antibody, including Fv antibodies comprised of the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. In some embodiments, the Fv antibody consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association, and the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In some embodiments, the six hypervariable regions confer antigen-binding specificity to the antibody. In some embodiments, a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen, including single domain antibodies isolated from camelid animals comprising one heavy chain variable domain such as VHH antibodies or nanobodies) has the ability to recognize and bind antigen. In some instances, the libraries disclosed herein comprise nucleic acids encoding for a scaffold, wherein the scaffold is a single-chain Fv or scFv, including antibody fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the desired structure for antigen binding. In some instances, a scFv is linked to the Fc fragment or a VHH is linked to the Fc fragment (including minibodies). In some instances, the antibody comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2) or subclass.

In some embodiments, libraries comprise immunoglobulins that are adapted to the species of an intended therapeutic target. Generally, these methods include “mammalization” and comprises methods for transferring donor antigen-binding information to a less immunogenic mammal antibody acceptor to generate useful therapeutic treatments. In some instances, the mammal is mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, and human. In some instances, provided herein are libraries and methods for felinization and caninization of antibodies.

“Humanized” forms of non-human antibodies can be chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. In some instances, these modifications are made to further refine antibody performance.

“Caninization” can comprise a method for transferring non-canine antigen-binding information from a donor antibody to a less immunogenic canine antibody acceptor to generate treatments useful as therapeutics in dogs. In some instances, caninized forms of non-canine antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-canine antibodies. In some instances, caninized antibodies are canine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the canine antibody are replaced by corresponding non-canine FR residues. In some instances, caninized antibodies include residues that are not found in the recipient antibody or in the donor antibody. In some instances, these modifications are made to further refine antibody performance. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a canine antibody.

“Felinization” can comprise a method for transferring non-feline antigen-binding information from a donor antibody to a less immunogenic feline antibody acceptor to generate treatments useful as therapeutics in cats. In some instances, felinized forms of non-feline antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-feline antibodies. In some instances, felinized antibodies are feline antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the feline antibody are replaced by corresponding non-feline FR residues. In some instances, felinized antibodies include residues that are not found in the recipient antibody or in the donor antibody. In some instances, these modifications are made to further refine antibody performance. The felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a felinize antibody.

Provided herein are libraries comprising nucleic acids encoding for a scaffold, wherein the scaffold is a non-immunoglobulin. In some instances, the scaffold is a non-immunoglobulin binding domain. For example, the scaffold is an antibody mimetic. Exemplary antibody mimetics include, but are not limited to, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, atrimers, DARPins, fynomers, Kunitz domain-based proteins, monobodies, anticalins, knottins, armadillo repeat protein-based proteins, and bicyclic peptides.

Libraries described herein comprising nucleic acids encoding for a scaffold, wherein the scaffold is an immunoglobulin, comprise variations in at least one region of the immunoglobulin. Exemplary regions of the antibody for variation include, but are not limited to, a complementarity-determining region (CDR), a variable domain, or a constant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. In some instances, the CDR is a heavy domain including, but not limited to, CDR-H1, CDR-H2, and CDR-H3. In some instances, the CDR is a light domain including, but not limited to, CDR-L1, CDR-L2, and CDR-L3. In some instances, the variable domain is variable domain, light chain (VL) or variable domain, heavy chain (VH). In some instances, the VL domain comprises kappa or lambda chains. In some instances, the constant domain is constant domain, light chain (CL) or constant domain, heavy chain (CH).

Methods described herein provide for synthesis of libraries comprising nucleic acids encoding for a scaffold, wherein each nucleic acid encodes for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the scaffold library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon of a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

In some instances, the at least one region of the immunoglobulin for variation is from heavy chain V-gene family, heavy chain D-gene family, heavy chain J-gene family, light chain V-gene family, or light chain J-gene family. See FIGS. 1A-1B. In some instances, the light chain V-gene family comprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL). Exemplary genes include, but are not limited to, IGHV1-18, IGHV1-69, IGHV1-8, IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV1-69, IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, and IGLV3-1. In some instances, the gene is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In some instances, the gene is IGHV1-69 and IGHV3-30. In some instances, the gene is IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2, or IGH1. In some instances, the gene is IGHJ3, IGHJ6, IGHJ, or IGHJ4.

Provided herein are libraries comprising nucleic acids encoding for immunoglobulin scaffolds, wherein the libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In some instances, the fragments comprise framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the scaffold libraries are synthesized with at least or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.

Libraries comprising nucleic acids encoding for immunoglobulin scaffolds as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the immunoglobulin scaffolds comprise at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino acids.

A number of variant sequences for the at least one region of the immunoglobulin for variation are de novo synthesized using methods as described herein. In some instances, a number of variant sequences is de novo synthesized for CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, VH, or combinations thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is at least or about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or more than 8000 sequences. In some instances, the number of variant sequences is about 10 to 500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150 to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325 sequences.

Variant sequences for the at least one region of the immunoglobulin, in some instances, vary in length or sequence. In some instances, the at least one region that is de novo synthesized is for CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, VH, or combinations thereof. In some instances, the at least one region that is de novo synthesized is for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 variant nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 additional nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 less nucleotides or amino acids as compared to wild-type. In some instances, the libraries comprise at least or about 10¹, 10², 10³ 10⁴ 10⁵ 10⁶, 10⁷ 10⁸, 10⁹ 10¹⁰ or more than 10¹⁰ variants.

Following synthesis of scaffold libraries, scaffold libraries may be used for screening and analysis. For example, scaffold libraries are assayed for library displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. In some instances, scaffold libraries are assayed by sequencing using various methods including, but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis.

In some instances, the scaffold libraries are assayed for functional activity, structural stability (e.g., thermal stable or pH stable), expression, specificity, or a combination thereof. In some instances, the scaffold libraries are assayed for scaffolds capable of folding. In some instances, a region of the antibody is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof. For example, a VH region or VL region is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof.

GLP1R Libraries

Provided herein are GLP1R binding libraries comprising nucleic acids encoding for scaffolds comprising sequences for GLP1R binding domains. In some instances, the scaffolds are immunoglobulins. In some instances, the scaffolds comprising sequences for GLP1R binding domains are determined by interactions between the GLP1R binding domains and the GLP1R.

Provided herein are libraries comprising nucleic acids encoding scaffolds comprising GLP1R binding domains, wherein the GLP1R binding domains are designed based on surface interactions on GLP1R. In some instances, the GLP1R comprises a sequence as defined by SEQ ID NO: 1. In some instances, the GLP1R binding domains interact with the amino- or carboxy-terminus of the GLP1R. In some instances, the GLP1R binding domains interact with at least one transmembrane domain including, but not limited to, transmembrane domain 1 (TM1), transmembrane domain 2 (TM2), transmembrane domain 3 (TM3), transmembrane domain 4 (TM4), transmembrane domain 5 (TM5), transmembrane domain 6 (TM6), and transmembrane domain 7 (TM7). In some instances, the GLP1R binding domains interact with an intracellular surface of the GLP1R. For example, the GLP1R binding domains interact with at least one intracellular loop including, but not limited to, intracellular loop 1 (ICL1), intracellular loop 2 (ICL2), and intracellular loop 3 (ICL3). In some instances, the GLP1R binding domains interact with an extracellular surface of the GLP1R. For example, the GLP1R binding domains interact with at least one extracellular domain (ECD) or extracellular loop (ECL) of the GLP1R. The extracellular loops include, but are not limited to, extracellular loop 1 (ECL1), extracellular loop 2 (ECL2), and extracellular loop 3 (ECL3).

Described herein are GLP1R binding domains, wherein the GLP1R binding domains are designed based on surface interactions between a GLP1R ligand and the GLP1R. In some instances, the ligand is a peptide. In some instances, the ligand is glucagon, glucagon-like peptide 1-(7-36) amide, glucagon-like peptide 1-(7-37), liraglutide, exendin-4, lixisenatide, T-0632, GLP1R0017, or BETP. In some instances, the ligand is a GLP1R agonist. In some instances, the ligand is a GLP1R antagonist. In some instances, the ligand is a GLP1R allosteric modulator. In some instances, the allosteric modulator is a negative allosteric modulator. In some instances, the allosteric modulator is a positive allosteric modulator.

Sequences of GLP1R binding domains based on surface interactions between a GLP1R ligand and the GLP1R are analyzed using various methods. For example, multispecies computational analysis is performed. In some instances, a structure analysis is performed. In some instances, a sequence analysis is performed. Sequence analysis can be performed using a database known in the art. Non-limiting examples of databases include, but are not limited to, NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi), UCSC Genome Browser (genome.ucsc.edu/), UniProt (www.uniprot.org/), and IUPHAR/BPS Guide to PHARMACOLOGY (guidetopharmacology.org/).

Described herein are GLP1R binding domains designed based on sequence analysis among various organisms. For example, sequence analysis is performed to identify homologous sequences in different organisms. Exemplary organisms include, but are not limited to, mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, fish, fly, and human.

Following identification of GLP1R binding domains, libraries comprising nucleic acids encoding for the GLP1R binding domains may be generated. In some instances, libraries of GLP1R binding domains comprise sequences of GLP1R binding domains designed based on conformational ligand interactions, peptide ligand interactions, small molecule ligand interactions, extracellular domains of GLP1R, or antibodies that target GLP1R. In some instances, libraries of GLP1R binding domains comprise sequences of GLP1R binding domains designed based on peptide ligand interactions. Libraries of GLP1R binding domains may be translated to generate protein libraries. In some instances, libraries of GLP1R binding domains are translated to generate peptide libraries, immunoglobulin libraries, derivatives thereof, or combinations thereof. In some instances, libraries of GLP1R binding domains are translated to generate protein libraries that are further modified to generate peptidomimetic libraries. In some instances, libraries of GLP1R binding domains are translated to generate protein libraries that are used to generate small molecules.

Methods described herein provide for synthesis of libraries of GLP1R binding domains comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the libraries of GLP1R binding domains comprise varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon in a GLP1R binding domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons in a GLP1R binding domain. An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of libraries comprising nucleic acids encoding for the GLP1R binding domains, wherein the libraries comprise sequences encoding for variation of length of the GLP1R binding domains. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less as compared to a predetermined reference sequence. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more than 300 codons more as compared to a predetermined reference sequence.

Following identification of GLP1R binding domains, the GLP1R binding domains may be placed in scaffolds as described herein. In some instances, the scaffolds are immunoglobulins. In some instances, the GLP1R binding domains are placed in the CDR-H3 region. GPCR binding domains that may be placed in scaffolds can also be referred to as a motif. Scaffolds comprising GLP1R binding domains may be designed based on binding, specificity, stability, expression, folding, or downstream activity. In some instances, the scaffolds comprising GLP1R binding domains enable contact with the GLP1R. In some instances, the scaffolds comprising GLP1R binding domains enables high affinity binding with the GLP1R. An exemplary amino acid sequence of GLP1R binding domain is described in Table 1.

TABLE 1 GLP1R amino acid sequences SEQ ID NO GPCR Amino Acid Sequence 1 GLP1R RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLF CNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGH VYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGERSSP EEQLLFLYIIYTVGYALSFSALVIASAILLGFRHLHCT RNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQ WDGLLSYQDSLSCRLVFLLMQYCVAANYYWLLVEGVYL YTLLAFSVLSEQWIFRLYVSIGWGVPLLFVVPWGIVKY LYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRV ICIVVSKLKANLMCKTDIKCRLAKSTLTLIPLLGTHEV IFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYC FVNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCPTS SLSSGATAGSSMYTATCQASCS

Provided herein are scaffolds comprising GLP1R binding domains, wherein the sequences of the GLP1R binding domains support interaction with GLP1R. The sequence may be homologous or identical to a sequence of a GLP1R ligand. In some instances, the GLP1R binding domain sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least or about 95% homology to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least or about 97% homology to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least or about 99% homology to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least or about 100% homology to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids of SEQ ID NO: 1.

The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

The term “homology” or “similarity” between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein sequence to the second protein sequence. Similarity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information).

Provided herein are GLP1R binding libraries comprising nucleic acids encoding for scaffolds comprising GLP1R binding domains comprise variation in domain type, domain length, or residue variation. In some instances, the domain is a region in the scaffold comprising the GLP1R binding domains. For example, the region is the VH, CDR-H3, or VL domain. In some instances, the domain is the GLP1R binding domain.

Methods described herein provide for synthesis of a GLP1R binding library of nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the GLP1R binding library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon of a VH, CDR-H3, or VL domain. In some instances, the variant library comprises sequences encoding for variation of at least a single codon in a GLP1R binding domain. For example, at least one single codon of a GLP1R binding domain as listed in Table 1 is varied. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a VH, CDR-H3, or VL domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons in a GLP1R binding domain. An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of a GLP1R binding library of nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence, wherein the GLP1R binding library comprises sequences encoding for variation of length of a domain. In some instances, the domain is VH, CDR-H3, or VL domain. In some instances, the domain is the GLP1R binding domain. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less as compared to a predetermined reference sequence. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more than 300 codons more as compared to a predetermined reference sequence.

Provided herein are GLP1R binding libraries comprising nucleic acids encoding for scaffolds comprising GLP1R binding domains, wherein the GLP1R binding libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the VH, CDR-H3, or VL domain. In some instances, the GLP1R binding libraries are synthesized with at least or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.

GLP1R binding libraries comprising nucleic acids encoding for scaffolds comprising GLP1R binding domains as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 to about 75 amino acids.

GLP1R binding libraries comprising de novo synthesized variant sequences encoding for scaffolds comprising GLP1R binding domains comprise a number of variant sequences. In some instances, a number of variant sequences is de novo synthesized for a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, VH, or a combination thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, a number of variant sequences is de novo synthesized for a GPCR binding domain. For example, the number of variant sequences is about 1 to about 10 sequences for the VH domain, about 10⁸ sequences for the GLP1R binding domain, and about 1 to about 44 sequences for the VK domain. The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is about 10 to 300, 25 to 275, 50 to 250, 75 to 225, 100 to 200, or 125 to 150 sequences.

GLP1R binding libraries comprising de novo synthesized variant sequences encoding for scaffolds comprising GLP1R binding domains comprise improved diversity. For example, variants are generated by placing GLP1R binding domain variants in immunoglobulin scaffold variants comprising N-terminal CDR-H3 variations and C-terminal CDR-H3 variations. In some instances, variants include affinity maturation variants. Alternatively or in combination, variants include variants in other regions of the immunoglobulin including, but not limited to, CDR-H1, CDR-H2, CDR-L1, CDR-L2, and CDR-L3. In some instances, the number of variants of the GLP1R binding libraries is least or about 10⁴ 10⁵ 10⁶, 10⁷ 10⁸, 10⁹ 10¹⁰ 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ non-identical sequences. For example, a library comprising about 10 variant sequences for a VH region, about 237 variant sequences for a CDR-H3 region, and about 43 variant sequences for a VL and CDR-L3 region comprises 10⁵ non-identical sequences (10×237×43).

Provided herein are libraries comprising nucleic acids encoding for a GLP1R antibody comprising variation in at least one region of the antibody, wherein the region is the CDR region. In some instances, the GLP1R antibody is a single domain antibody comprising one heavy chain variable domain such as a VHH antibody. In some instances, the VHH antibody comprises variation in one or more CDR regions. In some instances, libraries described herein comprise at least or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3. In some instances, libraries described herein comprise at least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰ or more than 10²⁰ sequences of a CDR1, CDR2, or CDR3. For example, the libraries comprise at least 2000 sequences of a CDR1, at least 1200 sequences for CDR2, and at least 1600 sequences for CDR3. In some instances, each sequence is non-identical.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain, light chain (VL). CDR1, CDR2, or CDR3 of a variable domain, light chain (VL) can be referred to as CDR-L1, CDR-L2, or CDR-L3, respectively. In some instances, libraries described herein comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3 of the VL. In some instances, libraries described herein comprise at least or about 10⁴ 10⁵ 10⁶, 10⁷ 10⁸, 10⁹ 10¹⁰ 10¹¹ 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences of a CDR1, CDR2, or CDR3 of the VL. For example, the libraries comprise at least 20 sequences of a CDR1 of the VL, at least 4 sequences of a CDR2 of the VL, and at least 140 sequences of a CDR3 of the VL. In some instances, the libraries comprise at least 2 sequences of a CDR1 of the VL, at least 1 sequence of CDR2 of the VL, and at least 3000 sequences of a CDR3 of the VL. In some instances, the VL is IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, or IGLV3-1. In some instances, the VL is IGKV2-28. In some instances, the VL is IGLV1-51.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain, heavy chain (VH). CDR1, CDR2, or CDR3 of a variable domain, heavy chain (VH) can be referred to as CDR-H1, CDR-H2, or CDR-H3, respectively. In some instances, libraries described herein comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3 of the VH. In some instances, libraries described herein comprise at least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences of a CDR1, CDR2, or CDR3 of the VH. For example, the libraries comprise at least 30 sequences of a CDR1 of the VH, at least 570 sequences of a CDR2 of the VH, and at least 10⁸ sequences of a CDR3 of the VH. In some instances, the libraries comprise at least 30 sequences of a CDR1 of the VH, at least 860 sequences of a CDR2 of the VH, and at least 10⁷ sequences of a CDR3 of the VH. In some instances, the VH is IGHV1-18, IGHV1 69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, or IGHV4-59/61. In some instances, the VH is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1 46, IGHV3-7, IGHV1, or IGHV1-8. In some instances, the VH is IGHV1-69 and IGHV3-30. In some instances, the VH is IGHV3-23.

Libraries as described herein, in some embodiments, comprise varying lengths of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3. In some instances, the length of the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or more than 90 amino acids in length. For example, the CDR-H3 comprises at least or about 12, 15, 16, 17, 20, 21, or 23 amino acids in length. In some instances, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 comprises a range of about 1 to about 10, about 5 to about 15, about 10 to about 20, or about 15 to about 30 amino acids in length.

Libraries comprising nucleic acids encoding for antibodies having variant CDR sequences as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the antibodies comprise at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino acids.

Ratios of the lengths of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 may vary in libraries described herein. In some instances, a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3 comprising at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or more than 90 amino acids in length comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the library. For example, a CDR-H3 comprising about 23 amino acids in length is present in the library at 40%, a CDR-H3 comprising about 21 amino acids in length is present in the library at 30%, a CDR-H3 comprising about 17 amino acids in length is present in the library at 20%, and a CDR-H3 comprising about 12 amino acids in length is present in the library at 10%. In some instances, a CDR-H3 comprising about 20 amino acids in length is present in the library at 40%, a CDR-H3 comprising about 16 amino acids in length is present in the library at 30%, a CDR-H3 comprising about 15 amino acids in length is present in the library at 20%, and a CDR-H3 comprising about 12 amino acids in length is present in the library at 10%.

Libraries as described herein encoding for a VHH antibody comprise variant CDR sequences that are shuffled to generate a library with a theoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences. In some instances, the library has a final library diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰ 10¹¹ 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences.

Provided herein are GLP1R binding libraries encoding for an immunoglobulin. In some instances, the GLP1R immunoglobulin is an antibody. In some instances, the GLP1R immunoglobulin is a VHH antibody. In some instances, the GLP1R immunoglobulin comprises a binding affinity (e.g., kD) to GLP1R of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 1 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 1.2 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 2 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 5 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 10 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 13.5 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 15 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 20 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 25 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 30 nM.

In some instances, the GLP1R immunoglobulin is a GLP1R agonist. In some instances, the GLP1R immunoglobulin is a GLP1R antagonist. In some instances, the GLP1R immunoglobulin is a GLP1R allosteric modulator. In some instances, the allosteric modulator is a negative allosteric modulator. In some instances, the allosteric modulator is a positive allosteric modulator. In some instances, the GLP1R immunoglobulin results in agonistic, antagonistic, or allosteric effects at a concentration of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, or more than 1000 nM. In some instances, the GLP1R immunoglobulin is a negative allosteric modulator. In some instances, the GLP1R immunoglobulin is a negative allosteric modulator at a concentration of at least or about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM. In some instances, the GLP1R immunoglobulin is a negative allosteric modulator at a concentration in a range of about 0.001 to about 100, 0.01 to about 90, about 0.1 to about 80, 1 to about 50, about 10 to about 40 nM, or about 1 to about 10 nM. In some instances, the GLP1R immunoglobulin comprises an EC50 or IC50 of at least or about 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.06, 0.07, 0.08, 0.9, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or more than 6 nM. In some instances, the GLP1R immunoglobulin comprises an EC50 or IC50 of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM.

Provided herein are GLP1R binding libraries encoding for an immunoglobulin, wherein the immunoglobulin comprises a long half-life. In some instances, the half-life of the GLP1R immunoglobulin is at least or about 12 hours, 24 hours 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 140 hours, 160 hours, 180 hours, 200 hours, or more than 200 hours. In some instances, the half-life of the GLP1R immunoglobulin is in a range of about 12 hours to about 300 hours, about 20 hours to about 280 hours, about 40 hours to about 240 hours, or about 60 hours to about 200 hours.

GLP1R immunoglobulins as described herein may comprise improved properties. In some instances, the GLP1R immunoglobulins are monomeric. In some instances, the GLP1R immunoglobulins are not prone to aggregation. In some instances, at least or about 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the GLP1R immunoglobulins are monomeric. In some instances, the GLP1R immunoglobulins are thermostable. In some instances, the GLP1R immunoglobulins result in reduced non-specific binding.

Following synthesis of GLP1R binding libraries comprising nucleic acids encoding scaffolds comprising GLP1R binding domains, libraries may be used for screening and analysis. For example, libraries are assayed for library displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. In some instances, the GLP1R binding libraries comprises nucleic acids encoding scaffolds comprising GPCR binding domains with multiple tags such as GFP, FLAG, and Lucy as well as a DNA barcode. In some instances, libraries are assayed by sequencing using various methods including, but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis.

Expression Systems

Provided herein are libraries comprising nucleic acids encoding for scaffolds comprising GLP1R binding domains, wherein the libraries have improved specificity, stability, expression, folding, or downstream activity. In some instances, libraries described herein are used for screening and analysis.

Provided herein are libraries comprising nucleic acids encoding for scaffolds comprising GLP1R binding domains, wherein the nucleic acid libraries are used for screening and analysis. In some instances, screening and analysis comprises in vitro, in vivo, or ex vivo assays. Cells for screening include primary cells taken from living subjects or cell lines. Cells may be from prokaryotes (e.g., bacteria and fungi) or eukaryotes (e.g., animals and plants). Exemplary animal cells include, without limitation, those from a mouse, rabbit, primate, and insect. In some instances, cells for screening include a cell line including, but not limited to, Chinese Hamster Ovary (CHO) cell line, human embryonic kidney (HEK) cell line, or baby hamster kidney (BHK) cell line. In some instances, nucleic acid libraries described herein may also be delivered to a multicellular organism. Exemplary multicellular organisms include, without limitation, a plant, a mouse, rabbit, primate, and insect.

Nucleic acid libraries or protein libraries encoded thereof described herein may be screened for various pharmacological or pharmacokinetic properties. In some instances, the libraries are screened using in vitro assays, in vivo assays, or ex vivo assays. For example, in vitro pharmacological or pharmacokinetic properties that are screened include, but are not limited to, binding affinity, binding specificity, and binding avidity. Exemplary in vivo pharmacological or pharmacokinetic properties of libraries described herein that are screened include, but are not limited to, therapeutic efficacy, activity, preclinical toxicity properties, clinical efficacy properties, clinical toxicity properties, immunogenicity, potency, and clinical safety properties.

Pharmacological or pharmacokinetic properties that may be screened include, but are not limited to, cell binding affinity and cell activity. For example, cell binding affinity assays or cell activity assays are performed to determine agonistic, antagonistic, or allosteric effects of libraries described herein. In some instances, the cell activity assay is a cAMP assay. In some instances, libraries as described herein are compared to cell binding or cell activity of ligands of GLP1R.

Libraries as described herein may be screened in cell based assays or in non-cell based assays. Examples of non-cell based assays include, but are not limited to, using viral particles, using in vitro translation proteins, and using protealiposomes with GLP1R.

Nucleic acid libraries as described herein may be screened by sequencing. In some instances, next generation sequence is used to determine sequence enrichment of GLP1R binding variants. In some instances, V gene distribution, J gene distribution, V gene family, CDR3 counts per length, or a combination thereof is determined. In some instances, clonal frequency, clonal accumulation, lineage accumulation, or a combination thereof is determined. In some instances, number of sequences, sequences with VH clones, clones, clones greater than 1, clonotypes, clonotypes greater than 1, lineages, simpsons, or a combination thereof is determined. In some instances, a percentage of non-identical CDR3s is determined. For example, the percentage of non identical CDR3s is calculated as the number of non-identical CDR3s in a sample divided by the total number of sequences that had a CDR3 in the sample.

Provided herein are nucleic acid libraries, wherein the nucleic acid libraries may be expressed in a vector. Expression vectors for inserting nucleic acid libraries disclosed herein may comprise eukaryotic or prokaryotic expression vectors. Exemplary expression vectors include, without limitation, mammalian expression vectors: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV—PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, (6His” disclosed as SEQ ID NO: 2410), pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-N1 Vector, pEFla-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV—PURO-NH2-CMYC; bacterial expression vectors: pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plant expression vectors: pRI 101-AN DNA and pCambia2301; and yeast expression vectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1/V5-His A and pDEST8. In some instances, the vector is pcDNA3 or pcDNA3.1.

Described herein are nucleic acid libraries that are expressed in a vector to generate a construct comprising a scaffold comprising sequences of GLP1R binding domains. In some instances, a size of the construct varies. In some instances, the construct comprises at least or about 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 bases. In some instances, a the construct comprises a range of about 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to 5,000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to 10,000, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 4,000, 1,000 to 5,000, 1,000 to 6,000, 1,000 to 7,000, 1,000 to 8,000, 1,000 to 9,000, 1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000 to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000, 3,000 to 8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000 to 5,000, 4,000 to 6,000, 4,000 to 7,000, 4,000 to 8,000, 4,000 to 9,000, 4,000 to 10,000, 5,000 to 6,000, 5,000 to 7,000, 5,000 to 8,000, 5,000 to 9,000, 5,000 to 10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 9,000, 6,000 to 10,000, 7,000 to 8,000, 7,000 to 9,000, 7,000 to 10,000, 8,000 to 9,000, 8,000 to 10,000, or 9,000 to 10,000 bases.

Provided herein are libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains, wherein the nucleic acid libraries are expressed in a cell. In some instances, the libraries are synthesized to express a reporter gene. Exemplary reporter genes include, but are not limited to, acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein, citrine fluorescent protein, orange fluorescent protein, cherry fluorescent protein, turquoise fluorescent protein, blue fluorescent protein, horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, and derivatives thereof. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), and antibiotic resistance determination.

Diseases and Disorders

Provided herein are GLP1R binding libraries comprising nucleic acids encoding for scaffolds comprising GLP1R binding domains that may have therapeutic effects. In some instances, the GLP1R binding libraries result in protein when translated that is used to treat a disease or disorder. In some instances, the protein is an immunoglobulin. In some instances, the protein is a peptidomimetic.

GLP1R libraries as described herein may comprise modulators of GLP1R. In some instances, the modulator of GLP1R is an inhibitor. In some instances, the modulator of GLP1R is an activator. In some instances, the GLP1R inhibitor is a GLP1R antagonist. In some instances, the GLP1R antagonist is GLP1R-3. In some instances, GLP1R-3 comprises SEQ ID NO: 2279. In some instances, GLP1R-3 comprises SEQ ID NO: 2320. Modulators of GLP1R, in some instances, are used for treating various diseases or disorders.

Exemplary diseases include, but are not limited to, cancer, inflammatory diseases or disorders, a metabolic disease or disorder, a cardiovascular disease or disorder, a respiratory disease or disorder, pain, a digestive disease or disorder, a reproductive disease or disorder, an endocrine disease or disorder, or a neurological disease or disorder. In some instances, the cancer is a solid cancer or a hematologic cancer. In some instances, a modulator of GLP1R as described herein is used for treatment of weight gain (or for inducing weight loss), treatment of obesity, or treatment of Type II diabetes. In some instances, the GLP1R modulator is used for treating hypoglycemia. In some instances, the GLP1R modulator is used for treating post-bariatric hypoglycemia. In some instances, the GLP1R modulator is used for treating severe hypoglycemia. In some instances, the GLP1R modulator is used for treating hyperinsulinism. In some instances, the GLP1R modulator is used for treating congenital hyperinsulinism.

In some instances, the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, or human. Subjects treated by methods described herein may be infants, adults, or children. Pharmaceutical compositions comprising antibodies or antibody fragments as described herein may be administered intravenously or subcutaneously.

Described herein are pharmaceutical compositions comprising antibodies or antibody fragment thereof that binds GLP1R. In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316. In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321; and wherein the immunoglobulin light chain comprises an amino acid sequence set forth in SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or 2316.

In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2303; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2310. In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2304; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2311. In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2305; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2312. In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2306; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2313. In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2307; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2314. In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2308; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2315. In some embodiments, the antibody or antibody fragment thereof comprises an immunoglobulin heavy chain and an immunoglobulin light chain: wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2309; and wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90%, 95%, 97%, 99%, or 100% identical to that set forth in SEQ ID NO: 2316.

In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDR-H3 comprising a sequence of any one of SEQ ID NOS: 2260-2276. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprise a sequence of any one of SEQ ID NOS: 2277-2295. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprise a sequence of any one of SEQ ID NOS: 2277, 2278, 2281, 2282, 2283, 2284, 2285, 2286, 2289, 2290, 2291, 2292, 2294, or 2295. In further instances, the pharmaceutical composition is used for treatment of a metabolic disorder.

Variant Libraries

Codon Variation

Variant nucleic acid libraries described herein may comprise a plurality of nucleic acids, wherein each nucleic acid encodes for a variant codon sequence compared to a reference nucleic acid sequence. In some instances, each nucleic acid of a first nucleic acid population contains a variant at a single variant site. In some instances, the first nucleic acid population contains a plurality of variants at a single variant site such that the first nucleic acid population contains more than one variant at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding multiple codon variants at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding up to 19 or more codons at the same position. The first nucleic acid population may comprise nucleic acids collectively encoding up to 60 variant triplets at the same position, or the first nucleic acid population may comprise nucleic acids collectively encoding up to 61 different triplets of codons at the same position. Each variant may encode for a codon that results in a different amino acid during translation. Table 3 provides a listing of each codon possible (and the representative amino acid) for a variant site.

TABLE 2 List of codons and amino acids One Three letter letter Amino Acids code code Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGC TGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAG Phenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine H His CAC CAT Isoleucine I Iso ATA ATC ATT Lysine K Lys AAA AAG Leucine L Leu TTA TTG CTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AAC AAT Proline P Pro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine T Thr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTT Tryptophan W Trp TGG Tyrosine Y Tyr TAC TAT

A nucleic acid population may comprise varied nucleic acids collectively encoding up to 20 codon variations at multiple positions. In such cases, each nucleic acid in the population comprises variation for codons at more than one position in the same nucleic acid. In some instances, each nucleic acid in the population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more codons in a single nucleic acid. In some instances, each variant long nucleic acid comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single long nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleic acid.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization, parallelization, and vertical integration of the end-to-end process from polynucleotide synthesis to gene assembly within nanowells on silicon to create a revolutionary synthesis platform. Devices described herein provide, with the same footprint as a 96-well plate, a silicon synthesis platform is capable of increasing throughput by a factor of up to 1,000 or more compared to traditional synthesis methods, with production of up to approximately 1,000,000 or more polynucleotides, or 10,000 or more genes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomic data has become an important factor for studies that delve into the biological roles of various genes in both normal biology and disease pathogenesis. At the core of this research is the central dogma of molecular biology and the concept of “residue-by-residue transfer of sequential information.” Genomic information encoded in the DNA is transcribed into a message that is then translated into the protein that is the active product within a given biological pathway.

Another exciting area of study is on the discovery, development and manufacturing of therapeutic molecules focused on a highly-specific cellular target. High diversity DNA sequence libraries are at the core of development pipelines for targeted therapeutics. Gene mutants are used to express proteins in a design, build, and test protein engineering cycle that ideally culminates in an optimized gene for high expression of a protein with high affinity for its therapeutic target. As an example, consider the binding pocket of a receptor. The ability to test all sequence permutations of all residues within the binding pocket simultaneously will allow for a thorough exploration, increasing chances of success. Saturation mutagenesis, in which a researcher attempts to generate all possible mutations at a specific site within the receptor, represents one approach to this development challenge. Though costly and time and labor-intensive, it enables each variant to be introduced into each position. In contrast, combinatorial mutagenesis, where a few selected positions or short stretch of DNA may be modified extensively, generates an incomplete repertoire of variants with biased representation.

To accelerate the drug development pipeline, a library with the desired variants available at the intended frequency in the right position available for testing—in other words, a precision library, enables reduced costs as well as turnaround time for screening. Provided herein are methods for synthesizing nucleic acid synthetic variant libraries which provide for precise introduction of each intended variant at the desired frequency. To the end user, this translates to the ability to not only thoroughly sample sequence space but also be able to query these hypotheses in an efficient manner, reducing cost and screening time. Genome-wide editing can elucidate important pathways, libraries where each variant and sequence permutation can be tested for optimal functionality, and thousands of genes can be used to reconstruct entire pathways and genomes to re-engineer biological systems for drug discovery.

In a first example, a drug itself can be optimized using methods described herein. For example, to improve a specified function of an antibody, a variant polynucleotide library encoding for a portion of the antibody is designed and synthesized. A variant nucleic acid library for the antibody can then be generated by processes described herein (e.g., PCR mutagenesis followed by insertion into a vector). The antibody is then expressed in a production cell line and screened for enhanced activity. Example screens include examining modulation in binding affinity to an antigen, stability, or effector function (e.g., ADCC, complement, or apoptosis). Exemplary regions to optimize the antibody include, without limitation, the Fc region, Fab region, variable region of the Fab region, constant region of the Fab region, variable domain of the heavy chain or light chain (VII or VL), and specific complementarity-determining regions (CDRs) of VII or VL.

Nucleic acid libraries synthesized by methods described herein may be expressed in various cells associated with a disease state. Cells associated with a disease state include cell lines, tissue samples, primary cells from a subject, cultured cells expanded from a subject, or cells in a model system. Exemplary model systems include, without limitation, plant and animal models of a disease state.

To identify a variant molecule associated with prevention, reduction or treatment of a disease state, a variant nucleic acid library described herein is expressed in a cell associated with a disease state, or one in which a cell a disease state can be induced. In some instances, an agent is used to induce a disease state in cells. Exemplary tools for disease state induction include, without limitation, a Cre/Lox recombination system, LPS inflammation induction, and streptozotocin to induce hypoglycemia. The cells associated with a disease state may be cells from a model system or cultured cells, as well as cells from a subject having a particular disease condition. Exemplary disease conditions include a bacterial, fungal, viral, autoimmune, or proliferative disorder (e.g., cancer). In some instances, the variant nucleic acid library is expressed in the model system, cell line, or primary cells derived from a subject, and screened for changes in at least one cellular activity. Exemplary cellular activities include, without limitation, proliferation, cycle progression, cell death, adhesion, migration, reproduction, cell signaling, energy production, oxygen utilization, metabolic activity, and aging, response to free radical damage, or any combination thereof.

Substrates

Devices used as a surface for polynucleotide synthesis may be in the form of substrates which include, without limitation, homogenous array surfaces, patterned array surfaces, channels, beads, gels, and the like. Provided herein are substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support the attachment and synthesis of polynucleotides. In some instances, substrates comprise a homogenous array surface. For example, the homogenous array surface is a homogenous plate. The term “locus” as used herein refers to a discrete region on a structure which provides support for polynucleotides encoding for a single predetermined sequence to extend from the surface. In some instances, a locus is on a two dimensional surface, e.g., a substantially planar surface. In some instances, a locus is on a three-dimensional surface, e.g., a well, microwell, channel, or post. In some instances, a surface of a locus comprises a material that is actively functionalized to attach to at least one nucleotide for polynucleotide synthesis, or preferably, a population of identical nucleotides for synthesis of a population of polynucleotides. In some instances, polynucleotide refers to a population of polynucleotides encoding for the same nucleic acid sequence. In some cases, a surface of a substrate is inclusive of one or a plurality of surfaces of a substrate. The average error rates for polynucleotides synthesized within a library described here using the systems and methods provided are often less than 1 in 1000, less than about 1 in 2000, less than about 1 in 3000 or less often without error correction.

Provided herein are surfaces that support the parallel synthesis of a plurality of polynucleotides having different predetermined sequences at addressable locations on a common support. In some instances, a substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides. In some cases, the surfaces provide support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more polynucleotides encoding for distinct sequences. In some instances, at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence. In some instances, the substrate provides a surface environment for the growth of polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.

Provided herein are methods for polynucleotide synthesis on distinct loci of a substrate, wherein each locus supports the synthesis of a population of polynucleotides. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus. In some instances, each polynucleotide sequence is synthesized with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more redundancy across different loci within the same cluster of loci on a surface for polynucleotide synthesis. In some instances, the loci of a substrate are located within a plurality of clusters. In some instances, a substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In some instances, a substrate comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or 10,000,000 or more distinct loci. In some instances, a substrate comprises about 10,000 distinct loci. The amount of loci within a single cluster is varied in different instances. In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances, each cluster includes about 50-500 loci. In some instances, each cluster includes about 100-200 loci. In some instances, each cluster includes about 100-150 loci. In some instances, each cluster includes about 109, 121, 130 or 137 loci. In some instances, each cluster includes about 19, 20, 61, 64 or more loci. Alternatively or in combination, polynucleotide synthesis occurs on a homogenous array surface.

In some instances, the number of distinct polynucleotides synthesized on a substrate is dependent on the number of distinct loci available in the substrate. In some instances, the density of loci within a cluster or surface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300, 400, 500, 1,000 or more loci per mm². In some cases, a substrate comprises 10 500, 25-400, 50-500, 100-500, 150-500, 10-250, 50-250, 10-200, or 50-200 mm². In some instances, the distance between the centers of two adjacent loci within a cluster or surface is from about 10-500, from about 10-200, or from about 10-100 um. In some instances, the distance between two centers of adjacent loci is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some instances, the distance between the centers of two adjacent loci is less than about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, each locus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some instances, the density of clusters within a substrate is at least or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 cluster per 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2 mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4 clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50 clusters per 1 mm² or more. In some instances, a substrate comprises from about 1 cluster per 10 mm² to about 10 clusters per 1 mm². In some instances, the distance between the centers of two adjacent clusters is at least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In some cases, the distance between the centers of two adjacent clusters is between about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In some cases, the distance between the centers of two adjacent clusters is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, each cluster has a cross section of about 0.5 to about 2, about 0.5 to about 1, or about 1 to about 2 mm. In some cases, each cluster has a cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interior cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

In some instances, a substrate is about the size of a standard 96 well plate, for example between about 100 and about 200 mm by between about 50 and about 150 mm. In some instances, a substrate has a diameter less than or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or 50 mm. In some instances, the diameter of a substrate is between about 25-1000, 25-800, 25 600, 25-500, 25-400, 25-300, or 25-200 mm. In some instances, a substrate has a planar surface area of at least about 100; 200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000; 40,000; 50,000 mm² or more. In some instances, the thickness of a substrate is between about 50 2000, 50-1000, 100-1000, 200-1000, or 250-1000 mm.

Surface Materials

Substrates, devices, and reactors provided herein are fabricated from any variety of materials suitable for the methods, compositions, and systems described herein. In certain instances, substrate materials are fabricated to exhibit a low level of nucleotide binding. In some instances, substrate materials are modified to generate distinct surfaces that exhibit a high level of nucleotide binding. In some instances, substrate materials are transparent to visible and/or UV light. In some instances, substrate materials are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of a substrate. In some instances, conductive materials are connected to an electric ground. In some instances, the substrate is heat conductive or insulated. In some instances, the materials are chemical resistant and heat resistant to support chemical or biochemical reactions, for example polynucleotide synthesis reaction processes. In some instances, a substrate comprises flexible materials. For flexible materials, materials can include, without limitation: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like. In some instances, a substrate comprises rigid materials. For rigid materials, materials can include, without limitation: glass; fuse silica; silicon, plastics (for example polytetraflouroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); metals (for example, gold, platinum, and the like). The substrate, solid support or reactors can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. The substrates/solid supports or the microstructures, reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.

Surface Architecture

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates have a surface architecture suitable for the methods, compositions, and systems described herein. In some instances, a substrate comprises raised and/or lowered features. One benefit of having such features is an increase in surface area to support polynucleotide synthesis. In some instances, a substrate having raised and/or lowered features is referred to as a three-dimensional substrate. In some cases, a three-dimensional substrate comprises one or more channels. In some cases, one or more loci comprise a channel. In some cases, the channels are accessible to reagent deposition via a deposition device such as a material deposition device. In some cases, reagents and/or fluids collect in a larger well in fluid communication one or more channels. For example, a substrate comprises a plurality of channels corresponding to a plurality of loci with a cluster, and the plurality of channels are in fluid communication with one well of the cluster. In some methods, a library of polynucleotides is synthesized in a plurality of loci of a cluster.

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates are configured for polynucleotide synthesis. In some instances, the structure is configured to allow for controlled flow and mass transfer paths for polynucleotide synthesis on a surface. In some instances, the configuration of a substrate allows for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during polynucleotide synthesis. In some instances, the configuration of a substrate allows for increased sweep efficiency, for example by providing sufficient volume for a growing polynucleotide such that the excluded volume by the growing polynucleotide does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the polynucleotide. In some instances, a three-dimensional structure allows for managed flow of fluid to allow for the rapid exchange of chemical exposure.

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates comprise structures suitable for the methods, compositions, and systems described herein. In some instances, segregation is achieved by physical structure. In some instances, segregation is achieved by differential functionalization of the surface generating active and passive regions for polynucleotide synthesis. In some instances, differential functionalization is achieved by alternating the hydrophobicity across the substrate surface, thereby creating water contact angle effects that cause beading or wetting of the deposited reagents. Employing larger structures can decrease splashing and cross-contamination of distinct polynucleotide synthesis locations with reagents of the neighboring spots. In some cases, a device, such as a material deposition device, is used to deposit reagents to distinct polynucleotide synthesis locations. Substrates having three-dimensional features are configured in a manner that allows for the synthesis of a large number of polynucleotides (e.g., more than about 10,000) with a low error rate (e.g., less than about 1:500, 1:1000, 1:1500, 1:2,000, 1:3,000, 1:5,000, or 1:10,000). In some cases, a substrate comprises features with a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm².

A well of a substrate may have the same or different width, height, and/or volume as another well of the substrate. A channel of a substrate may have the same or different width, height, and/or volume as another channel of the substrate. In some instances, the diameter of a cluster or the diameter of a well comprising a cluster, or both, is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some instances, the diameter of a cluster or well or both is less than or about 5, 4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05 mm. In some instances, the diameter of a cluster or well or both is between about 1.0 and 1.3 mm. In some instances, the diameter of a cluster or well, or both is about 1.150 mm. In some instances, the diameter of a cluster or well, or both is about 0.08 mm. The diameter of a cluster refers to clusters within a two-dimensional or three-dimensional substrate.

In some instances, the height of a well is from about 20-1000, 50-1000, 100-1000, 200 1000, 300-1000, 400-1000, or 500-1000 um. In some cases, the height of a well is less than about 1000, 900, 800, 700, or 600 um.

In some instances, a substrate comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of a channel is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, or 10-50 um. In some cases, the height of a channel is less than 100, 80, 60, 40, or 20 um.

In some instances, the diameter of a channel, locus (e.g., in a substantially planar substrate) or both channel and locus (e.g., in a three-dimensional substrate wherein a locus corresponds to a channel) is from about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, for example, about 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the diameter of a channel, locus, or both channel and locus is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the distance between the center of two adjacent channels, loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200, 5-100, 5-50, or 5-30, for example, about 20 um.

Surface Modifications

Provided herein are methods for polynucleotide synthesis on a surface, wherein the surface comprises various surface modifications. In some instances, the surface modifications are employed for the chemical and/or physical alteration of a surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of a substrate surface. For example, surface modifications include, without limitation, (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e., providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e., removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.

In some cases, the addition of a chemical layer on top of a surface (referred to as adhesion promoter) facilitates structured patterning of loci on a surface of a substrate. Exemplary surfaces for application of adhesion promotion include, without limitation, glass, silicon, silicon dioxide and silicon nitride. In some cases, the adhesion promoter is a chemical with a high surface energy. In some instances, a second chemical layer is deposited on a surface of a substrate. In some cases, the second chemical layer has a low surface energy. In some cases, surface energy of a chemical layer coated on a surface supports localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci are alterable.

In some instances, a substrate surface, or resolved loci, onto which nucleic acids or other moieties are deposited, e.g., for polynucleotide synthesis, are smooth or substantially planar (e.g., two-dimensional) or have irregularities, such as raised or lowered features (e.g., three-dimensional features). In some instances, a substrate surface is modified with one or more different layers of compounds. Such modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like.

In some instances, resolved loci of a substrate are functionalized with one or more moieties that increase and/or decrease surface energy. In some cases, a moiety is chemically inert. In some cases, a moiety is configured to support a desired chemical reaction, for example, one or more processes in a polynucleotide synthesis reaction. The surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface. In some instances, a method for substrate functionalization comprises: (a) providing a substrate having a surface that comprises silicon dioxide; and (b) silanizing the surface using, a suitable silanizing agent described herein or otherwise known in the art, for example, an organofunctional alkoxysilane molecule. Methods and functionalizing agents are described in U.S. Pat. No. 5,474,796, which is herein incorporated by reference in its entirety.

In some instances, a substrate surface is functionalized by contact with a derivatizing composition that contains a mixture of silanes, under reaction conditions effective to couple the silanes to the substrate surface, typically via reactive hydrophilic moieties present on the substrate surface. Silanization generally covers a surface through self-assembly with organofunctional alkoxysilane molecules. A variety of siloxane functionalizing reagents can further be used as currently known in the art, e.g., for lowering or increasing surface energy. The organofunctional alkoxysilanes are classified according to their organic functions.

Polynucleotide Synthesis

Methods of the current disclosure for polynucleotide synthesis may include processes involving phosphoramidite chemistry. In some instances, polynucleotide synthesis comprises coupling a base with phosphoramidite. Polynucleotide synthesis may comprise coupling a base by deposition of phosphoramidite under coupling conditions, wherein the same base is optionally deposited with phosphoramidite more than once, i.e., double coupling. Polynucleotide synthesis may comprise capping of unreacted sites. In some instances, capping is optional. Polynucleotide synthesis may also comprise oxidation or an oxidation step or oxidation steps. Polynucleotide synthesis may comprise deblocking, detritylation, and sulfurization. In some instances, polynucleotide synthesis comprises either oxidation or sulfurization. In some instances, between one or each step during a polynucleotide synthesis reaction, the device is washed, for example, using tetrazole or acetonitrile. Time frames for any one step in a phosphoramidite synthesis method may be less than about 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Polynucleotide synthesis using a phosphoramidite method may comprise a subsequent addition of a phosphoramidite building block (e.g., nucleoside phosphoramidite) to a growing polynucleotide chain for the formation of a phosphite triester linkage. Phosphoramidite polynucleotide synthesis proceeds in the 3′ to 5′ direction. Phosphoramidite polynucleotide synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid chain per synthesis cycle. In some instances, each synthesis cycle comprises a coupling step. Phosphoramidite coupling involves the formation of a phosphite triester linkage between an activated nucleoside phosphoramidite and a nucleoside bound to the substrate, for example, via a linker. In some instances, the nucleoside phosphoramidite is provided to the device activated. In some instances, the nucleoside phosphoramidite is provided to the device with an activator. In some instances, nucleoside phosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over the substrate-bound nucleosides. In some instances, the addition of nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile. Following addition of a nucleoside phosphoramidite, the device is optionally washed. In some instances, the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate. In some instances, a polynucleotide synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps. Prior to coupling, in many cases, the nucleoside bound to the device is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization. A common protecting group is 4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methods optionally comprise a capping step. In a capping step, the growing polynucleotide is treated with a capping agent. A capping step is useful to block unreacted substrate-bound 5′-OH groups after coupling from further chain elongation, preventing the formation of polynucleotides with internal base deletions. Further, phosphoramidites activated with 1H-tetrazole may react, to a small extent, with the O6 position of guanosine. Without being bound by theory, upon oxidation with I2/water, this side product, possibly via O6-N7 migration, may undergo depurination. The apurinic sites may end up being cleaved in the course of the final deprotection of the polynucleotide thus reducing the yield of the full-length product. The O6 modifications may be removed by treatment with the capping reagent prior to oxidation with 12/water. In some instances, inclusion of a capping step during polynucleotide synthesis decreases the error rate as compared to synthesis without capping. As an example, the capping step comprises treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1-methylimidazole. Following a capping step, the device is optionally washed.

In some instances, following addition of a nucleoside phosphoramidite, and optionally after capping and one or more wash steps, the device bound growing nucleic acid is oxidized. The oxidation step comprises the phosphite triester is oxidized into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleoside linkage. In some instances, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base (e.g., pyridine, lutidine, collidine). Oxidation may be carried out under anhydrous conditions using, e.g. tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, a capping step is performed following oxidation. A second capping step allows for device drying, as residual water from oxidation that may persist can inhibit subsequent coupling. Following oxidation, the device and growing polynucleotide is optionally washed. In some instances, the step of oxidation is substituted with a sulfurization step to obtain polynucleotide phosphorothioates, wherein any capping steps can be performed after the sulfurization. Many reagents are capable of the efficient sulfur transfer, including but not limited to 3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent, and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occur through coupling, the protected 5′ end of the device bound growing polynucleotide is removed so that the primary hydroxyl group is reactive with a next nucleoside phosphoramidite. In some instances, the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound polynucleotide and thus reduces the yield of the desired full-length product. Methods and compositions of the disclosure described herein provide for controlled deblocking conditions limiting undesired depurination reactions. In some instances, the device bound polynucleotide is washed after deblocking. In some instances, efficient washing after deblocking contributes to synthesized polynucleotides having a low error rate.

Methods for the synthesis of polynucleotides typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g., locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it is reactive with a subsequently applied protected monomer; and application of another protected monomer for linking. One or more intermediate steps include oxidation or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.

Methods for phosphoramidite-based polynucleotide synthesis comprise a series of chemical steps. In some instances, one or more steps of a synthesis method involve reagent cycling, where one or more steps of the method comprise application to the device of a reagent useful for the step. For example, reagents are cycled by a series of liquid deposition and vacuum drying steps. For substrates comprising three-dimensional features such as wells, microwells, channels and the like, reagents are optionally passed through one or more regions of the device via the wells and/or channels.

Methods and systems described herein relate to polynucleotide synthesis devices for the synthesis of polynucleotides. The synthesis may be in parallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or more polynucleotides can be synthesized in parallel. The total number polynucleotides that may be synthesized in parallel may be from 2-100000, 3-50000, 4 10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700, 11-650, 12-600, 13-550, 14-500, 15-450, 16 400, 17-350, 18-300, 19-250, 20-200, 21-150, 22-100, 23-50, 24-45, 25-40, 30-35. Those of skill in the art appreciate that the total number of polynucleotides synthesized in parallel may fall within any range bound by any of these values, for example 25-100. The total number of polynucleotides synthesized in parallel may fall within any range defined by any of the values serving as endpoints of the range. Total molar mass of polynucleotides synthesized within the device or the molar mass of each of the polynucleotides may be at least or at least about 10, 20, 30, 40, 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at least or about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 300, 400, 500 nucleotides, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at most or about at most 500, 400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 nucleotides, or less. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100, 15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciate that the length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range bound by any of these values, for example 100-300. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range defined by any of the values serving as endpoints of the range.

Methods for polynucleotide synthesis on a surface provided herein allow for synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotides per hour, or more are synthesized. Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified versions thereof. In some instances, libraries of polynucleotides are synthesized in parallel on substrate. For example, a device comprising about or at least about 100; 1,000; 10,000; 30,000; 75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or 5,000,000 resolved loci is able to support the synthesis of at least the same number of distinct polynucleotides, wherein polynucleotide encoding a distinct sequence is synthesized on a resolved locus. In some instances, a library of polynucleotides is synthesized on a device with low error rates described herein in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less. In some instances, larger nucleic acids assembled from a polynucleotide library synthesized with low error rate using the substrates and methods described herein are prepared in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.

In some instances, methods described herein provide for generation of a library of nucleic acids comprising variant nucleic acids differing at a plurality of codon sites. In some instances, a nucleic acid may have 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16 sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50 sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may be adjacent. In some instances, the one or more sites of variant codon sites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more codons.

In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein all the variant codon sites are adjacent to one another, forming a stretch of variant codon sites. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein none the variant codon sites are adjacent to one another. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein some the variant codon sites are adjacent to one another, forming a stretch of variant codon sites, and some of the variant codon sites are not adjacent to one another.

Referring to the Figures, FIG. 3 illustrates an exemplary process workflow for synthesis of nucleic acids (e.g., genes) from shorter nucleic acids. The workflow is divided generally into phases: (1) de novo synthesis of a single stranded nucleic acid library, (2) joining nucleic acids to form larger fragments, (3) error correction, (4) quality control, and (5) shipment. Prior to de novo synthesis, an intended nucleic acid sequence or group of nucleic acid sequences is preselected. For example, a group of genes is preselected for generation.

Once large nucleic acids for generation are selected, a predetermined library of nucleic acids is designed for de novo synthesis. Various suitable methods are known for generating high density polynucleotide arrays. In the workflow example, a device surface layer is provided. In the example, chemistry of the surface is altered in order to improve the polynucleotide synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy are generated to attract liquids. The surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions or microwells which increase surface area. In the workflow example, high surface energy molecules selected serve a dual function of supporting DNA chemistry, as disclosed in International Patent Application Publication WO/2015/021080, which is herein incorporated by reference in its entirety.

In situ preparation of polynucleotide arrays is generated on a solid support and utilizes single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 302. In some instances, polynucleotides are cleaved from the surface at this stage. Cleavage includes gas cleavage, e.g., with ammonia or methylamine.

The generated polynucleotide libraries are placed in a reaction chamber. In this exemplary workflow, the reaction chamber (also referred to as “nanoreactor”) is a silicon coated well, containing PCR reagents and lowered onto the polynucleotide library 303. Prior to or after the sealing 304 of the polynucleotides, a reagent is added to release the polynucleotides from the substrate. In the exemplary workflow, the polynucleotides are released subsequent to sealing of the nanoreactor 305. Once released, fragments of single stranded polynucleotides hybridize in order to span an entire long range sequence of DNA. Partial hybridization 305 is possible because each synthesized polynucleotide is designed to have a small portion overlapping with at least one other polynucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerase cycles, the polynucleotides anneal to complementary fragments and gaps are filled in by a polymerase. Each cycle increases the length of various fragments randomly depending on which polynucleotides find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA 306.

After PCA is complete, the nanoreactor is separated from the device 307 and positioned for interaction with a device having primers for PCR 308. After sealing, the nanoreactor is subject to PCR 309 and the larger nucleic acids are amplified. After PCR 310, the nanochamber is opened 311, error correction reagents are added 312, the chamber is sealed 313 and an error correction reaction occurs to remove mismatched base pairs and/or strands with poor complementarity from the double stranded PCR amplification products 314. The nanoreactor is opened and separated 315. Error corrected product is next subject to additional processing steps, such as PCR and molecular bar coding, and then packaged 322 for shipment 323.

In some instances, quality control measures are taken. After error correction, quality control steps include for example interaction with a wafer having sequencing primers for amplification of the error corrected product 316, sealing the wafer to a chamber containing error corrected amplification product 317, and performing an additional round of amplification 318. The nanoreactor is opened 319 and the products are pooled 320 and sequenced 321. After an acceptable quality control determination is made, the packaged product 322 is approved for shipment 323.

In some instances, a nucleic acid generated by a workflow such as that in FIG. 3 is subject to mutagenesis using overlapping primers disclosed herein. In some instances, a library of primers are generated by in situ preparation on a solid support and utilize single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 302.

Computer Systems

Any of the systems described herein, may be operably linked to a computer and may be automated through a computer either locally or remotely. In various instances, the methods and systems of the disclosure may further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the disclosure. The computer systems may be programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.

The computer system 400 illustrated in FIG. 4 may be understood as a logical apparatus that can read instructions from media 411 and/or a network port 405, which can optionally be connected to server 409 having fixed media 412. The system, such as shown in FIG. 4 can include a CPU 401, disk drives 403, optional input devices such as keyboard 415 and/or mouse 416 and optional monitor 407. Data communication can be achieved through the indicated communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting and/or receiving data. For example, the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception and/or review by a party 422 as illustrated in FIG. 4.

FIG. 5 is a block diagram illustrating a first example architecture of a computer system 500 that can be used in connection with example instances of the present disclosure. As depicted in FIG. 5, the example computer system can include a processor 502 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100TM processor, ARM Cortex-A8 Apple A4™ processor, Marvell PXA 930™ processor, or a functionally-equivalent processor. Multiple threads of execution can be used for parallel processing. In some instances, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, and/or personal data assistant devices.

As illustrated in FIG. 5, a high speed cache 504 can be connected to, or incorporated in, the processor 502 to provide a high speed memory for instructions or data that have been recently, or are frequently, used by the processor 502. The processor 502 is connected to a north bridge 506 by a processor bus 508. The north bridge 506 is connected to random access memory (RAM) 510 by a memory bus 512 and manages access to the RAM 510 by the processor 502. The north bridge 506 is also connected to a south bridge 514 by a chipset bus 516. The south bridge 514 is, in turn, connected to a peripheral bus 518. The peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are often referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus 518. In some alternative architectures, the functionality of the north bridge can be incorporated into the processor instead of using a separate north bridge chip. In some instances, system 500 can include an accelerator card 522 attached to the peripheral bus 518. The accelerator can include field programmable gate arrays (FPGAs) or other hardware for accelerating certain processing. For example, an accelerator can be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.

Software and data are stored in external storage 524 and can be loaded into RAM 510 and/or cache 504 for use by the processor. The system 500 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalent operating systems, as well as application software running on top of the operating system for managing data storage and optimization in accordance with example instances of the present disclosure. In this example, system 500 also includes network interface cards (NICs) 520 and 521 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.

FIG. 6 is a diagram showing a network 600 with a plurality of computer systems 602 a, and 602 b, a plurality of cell phones and personal data assistants 602 c, and Network Attached Storage (NAS) 604 a, and 604 b. In example instances, systems 602 a, 602 b, and 602 c can manage data storage and optimize data access for data stored in Network Attached Storage (NAS) 604 a and 604 b. A mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems 602 a, and 602 b, and cell phone and personal data assistant systems 602 c. Computer systems 602 a, and 602 b, and cell phone and personal data assistant systems 602 c can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS) 604 a and 604 b. FIG. 6 illustrates an example only, and a wide variety of other computer architectures and systems can be used in conjunction with the various instances of the present disclosure. For example, a blade server can be used to provide parallel processing. Processor blades can be connected through a back plane to provide parallel processing. Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface. In some example instances, processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors. In other instances, some or all of the processors can use a shared virtual address memory space.

FIG. 7 is a block diagram of a multiprocessor computer system 700 using a shared virtual address memory space in accordance with an example instance. The system includes a plurality of processors 702 a-f that can access a shared memory subsystem 704. The system incorporates a plurality of programmable hardware memory algorithm processors (MAPs) 706 a-f in the memory subsystem 704. Each MAP 706 a-f can comprise a memory 708 a-f and one or more field programmable gate arrays (FPGAs) 710 a-f. The MAP provides a configurable functional unit and particular algorithms or portions of algorithms can be provided to the FPGAs 710 a-f for processing in close coordination with a respective processor. For example, the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example instances. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access an associated memory 708 a-f, allowing it to execute tasks independently of, and asynchronously from the respective microprocessor 702 a-f. In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example instances, including systems using any combination of general processors, co-processors, FPGAs and other programmable logic devices, system on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some instances, all or part of the computer system can be implemented in software or hardware. Any variety of data storage media can be used in connection with example instances, including random access memory, hard drives, flash memory, tape drives, disk arrays, Network Attached Storage (NAS) and other local or distributed data storage devices and systems.

In example instances, the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems. In other instances, the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs) as referenced in FIG. 5, system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card, such as accelerator card 522 illustrated in FIG. 5.

The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of a library of polynucleotides. The device surface was first wet cleaned using a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20 minutes. The device was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min, and dried with Nz. The device was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI water for 1 min each, and then rinsed again with DI water using the handgun. The device was then plasma cleaned by exposing the device surface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at 250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solution comprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface was resist coated using a Brewer Science 200× spin coater. SPR™ 3612 photoresist was spin coated on the device at 2500 rpm for 40 sec. The device was pre-baked for 30 min at 90° C. on a Brewer hot plate. The device was subjected to photolithography using a Karl Suss MA6 mask aligner instrument. The device was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the device soaked in water for 5 min. The device was baked for 30 min at 100° C. in the oven, followed by visual inspection for lithography defects using a Nikon L200. A descum process was used to remove residual resist using the SAMCO PC-300 instrument to O₂ plasma etch at 250 watts for 1 min.

The device surface was passively functionalized with a 100 μL solution of perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. The device was placed in a chamber, pumped for 10 min, and then the valve was closed to the pump and left to stand for 10 min. The chamber was vented to air. The device was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power (9 on Crest system). The device was then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power. The device was dipped in 300 mL of 200 proof ethanol and blown dry with Nz. The functionalized surface was activated to serve as a support for polynucleotide synthesis.

Example 2: Synthesis of a 50-Mer Sequence on an Oligonucleotide Synthesis Device

A two dimensional oligonucleotide synthesis device was assembled into a flowcell, which was connected to a flowcell (Applied Biosystems (ABI394 DNA Synthesizer”). The two-dimensional oligonucleotide synthesis device was uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used to synthesize an exemplary polynucleotide of 50 bp (“50-mer polynucleotide”) using polynucleotide synthesis methods described herein.

The sequence of the 50-mer was as described in SEQ ID NO.: 2. 5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT ##TTTTTTT TTT3′ (SEQ ID NO.: 2), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linker enabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling, capping, oxidation, and deblocking) according to the protocol in Table 3 and an ABI synthesizer.

TABLE 3 Synthesis protocols General DNA Synthesis Table 3 Process Name Process Step Time (sec) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 23 N2 System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION Activator Manifold Flush 2 (Phosphoramidite + Activator to Flowcell 6 Activator Flow) Activator + 6 Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5 Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5 Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5 Phosphoramidite to Flowcell Incubate for 25sec 25 WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION Activator Manifold Flush 2 (Phosphoramidite + Activator to Flowcell 5 Activator Flow) Activator + 18 Phosphoramidite to Flowcell Incubate for 25sec 25 WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 CAPPING (CapA + B, CapA + B to Flowcell 15 1:1, Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 Acetonitrile System Flush 4 OXIDATION (Oxidizer Oxidizer to Flowcell 18 Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 System Flush 4 Acetonitrile System Flush 4 DEBLOCKING (Deblock Deblock to Flowcell 36 Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 18 N2 System Flush 4.13 Acetonitrile System Flush 4.13 Acetonitrile to Flowcell 15

The phosphoramidite/activator combination was delivered similar to the delivery of bulk reagents through the flowcell. No drying steps were performed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enable faster flow. Without flow restrictor, flow rates for amidites (0.1M in ACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx from GlenResearch) in ACN), and Ox (0.02M 12 in 20% pyridine, 10% water, and 70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and capping reagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride in THF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200 uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly ˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flow restrictor). The time to completely push out Oxidizer was observed, the timing for chemical flow times was adjusted accordingly and an extra ACN wash was introduced between different chemicals. After polynucleotide synthesis, the chip was deprotected in gaseous ammonia overnight at 75 psi. Five drops of water were applied to the surface to recover polynucleotides. The recovered polynucleotides were then analyzed on a BioAnalyzer small RNA chip.

Example 3: Synthesis of a 100-Mer Sequence on an Oligonucleotide Synthesis Device

The same process as described in Example 2 for the synthesis of the 50-mer sequence was used for the synthesis of a 100-mer polynucleotide (“100-mer polynucleotide”; 5′ CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATG CTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT ##TTTTTTTTTT3′, where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes); SEQ ID NO.: 3) on two different silicon chips, the first one uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second one functionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane, and the polynucleotides extracted from the surface were analyzed on a BioAnalyzer instrument.

All ten samples from the two chips were further PCR amplified using a forward (5′ATGCGGGGTTCTCATCATC3; SEQ ID NO.: 4) and a reverse (5′CGGGATCCTTATCGTCATCG3; SEQ ID NO.: 5) primer in a 50 uL PCR mix (25 uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverse primer, 1 uL polynucleotide extracted from the surface, and water up to 50 uL) using the following thermalcycling program: 98° C., 30 sec 98° C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles 72° C., 2 min

The PCR products were also run on a BioAnalyzer, demonstrating sharp peaks at the 100-mer position. Next, the PCR amplified samples were cloned, and Sanger sequenced. Table 4 summarizes the results from the Sanger sequencing for samples taken from spots 1-5 from chip 1 and for samples taken from spots 6-10 from chip 2.

TABLE 4 Sequencing results Spot Error rate Cycle efficiency  1 1/763 bp 99.87%  2 1/824 bp 99.88%  3 1/780 bp 99.87%  4 1/429 bp 99.77%  5 1/1525 bp  99.93%  6 1/1615 bp  99.94%  7 1/531 bp 99.81%  8 1/1769 bp  99.94%  9 1/854 bp 99.88% 10 1/1451 bp  99.93%

Thus, the high quality and uniformity of the synthesized polynucleotides were repeated on two chips with different surface chemistries. Overall, 89% of the 100-mers that were sequenced were perfect sequences with no errors, corresponding to 233 out of 262.

Table 5 summarizes error characteristics for the sequences obtained from the polynucleotide samples from spots 1-10.

TABLE 5 Error characteristics Sample ID/ Spot no. OSA_0046/1 OSA_0047/2 OSA_0048/3 OSA_0049/4 OSA_0050/5 OSA_0051/6 Total Sequences 32 32 32 32 32 32 Sequencing 25 of 28 27 of 27 26 of 30 21 of 23 25 of 26 29 of 30 Quality Oligo Quality 23 of 25 25 of 27 22 of 26 18 of 21 24 of 25 25 of 29 ROI Match Count 2500 2698 2561 2122 2499 2666 ROI Mutation 2 2 1 3 1 0 ROI Multi Base 0 0 0 0 0 0 Deletion ROI Small 1 0 0 0 0 0 Insertion ROI Single 0 0 0 0 0 0 Base Deletion Large Deletion 0 0 1 0 0 1 Count Mutation: G > A 2 2 1 2 1 0 Mutation: T > C 0 0 0 1 0 0 ROI Error Count 3 2 2 3 1 1 ROI Error Rate Err: ~1 in Err: ~1 in Err: ~1 in Err: ~1 in Err: ~1 in Err: ~1 in 834 1350 1282 708 2500 2667 ROI Minus Primer MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 Error Rate in 763 in 824 in 780 in 429 in 1525 in 1615 Sample ID/ Spot no. OSA_0052/7 OSA_0053/8 OSA_0054/9 OSA_0055/10 Total Sequences 32 32 32 32 Sequencing Quality 27 of 31 29 of 31 28 of 29 25 of 28 Oligo Quality 22 of 27 28 of 29 26 of 28 20 of 25 ROI Match Count 2625 2899 2798 2348 ROI Mutation 2 1 2 1 ROI Multi Base 0 0 0 0 Deletion ROI Small 0 0 0 0 Insertion ROI Single Base 0 0 0 0 Deletion Large Deletion 1 0 0 0 Count Mutation: G > A 2 1 2 1 Mutation: T > C 0 0 0 0 ROI Error Count 3 1 2 1 ROI Error Rate Err: ~1 in 876 Err: ~1 in 2900 Err: ~1 in 1400 Err: ~1 in 2349 ROI Minus Primer MP Err: ~1 in MP Err: ~1 in MP Err: ~1 in MP Err: ~1 in Error Rate 531 1769 854 1451

Example 4: Design of GLP1R Binding Domains Based on Peptide Ligand Interactions

GLP1R binding domains were designed based on interaction surfaces between peptide ligands that interact with GLP1R. Motif variants were generated based on the interaction surface of the peptides with the ECD as well as with the N-terminal GLP1R ligand interaction surface. This was done using structural modeling. Exemplary motif variants were created based on glucagon like peptide's interaction with GLP1R as seen in Table 6. The motif variant sequences were generated using the following sequence from glucagon like peptide:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. (SEQ ID NO: 6)

TABLE 6 Variant amino acid sequences for glucagon like peptide SEQ ID NO. Variant Amino Acid Sequence  7 1 sggggsggggsggggHAEGTFTSDVSSYLEGQAA KEFIAWLV  8 2 sggggsggggsggggAEGTFTSDVSSYLEGQAAK EFIAWLV  9 3 sggggsggggsggggEGTFTSDVSSYLEGQAAKE FIAWLV 10 4 sggggsggggsggggGTFTSDVSSYLEGQAAKEF IAWLV 11 5 sggggsggggsggggTFTSDVSSYLEGQAAKEFI AWLV 12 6 sggggsggggsggggFTSDVSSYLEGQAAKEFIA WLV 13 7 sggggsggggsggggTSDVSSYLEGQAAKEFIAW LV 14 8 sggggsggggsggggSDVSSYLEGQAAKEFIAWL V 15 9 sggggsggggsggggDVSSYLEGQAAKEFIAWLV

Example 5: Design of Antibody Scaffolds

To generate scaffolds, structural analysis, repertoire sequencing analysis of the heavy chain, and specific analysis of heterodimer high-throughput sequencing datasets were performed. Each heavy chain was associated with each light chain scaffold. Each heavy chain scaffold was assigned 5 different long CDR-H3 loop options. Each light chain scaffold was assigned 5 different L3 scaffolds. The heavy chain CDR-H3 stems were chosen from the frequently observed long H3 loop stems (10 amino acids on the N-terminus and the C-terminus) found both across individuals and across V-gene segments. The light chain scaffold L3s were chosen from heterodimers comprising long H3s. Direct heterodimers based on information from the Protein Data Bank (PDB) and deep sequencing datasets were used in which CDR H1, H2, L1, L2, L3, and CDR-H3 stems were fixed. The various scaffolds were then formatted for display on phage to assess for expression.

Structural Analysis

About 2,017 antibody structures were analyzed from which 22 structures with long CDR-H3s of at least 25 amino acids in length were observed. The heavy chains included the following: IGHV1-69, IGHV3-30, IGHV4-49, and IGHV3-21. The light chains identified included the following: IGLV3-21, IGKV3-11, IGKV2-28, IGKV1-5, IGLV1-51, IGLV1-44, and IGKV1-13. In the analysis, four heterodimer combinations were observed multiple times including: IGHV4-59/61-IGLV3-21, IGHV3-21-IGKV2-28, IGHV1-69-IGKV3-11, and IGHV1-69-IGKV1-5. An analysis of sequences and structures identified intra-CDR-H3 disulfide bonds in a few structures with packing of bulky side chains such as tyrosine in the stem providing support for long H₃ stability. Secondary structures including beta-turn-beta sheets and a “hammerhead” subdomain were also observed.

Repertoire Analysis

A repertoire analysis was performed on 1,083,875 IgM+/CD27-naive B cell receptor (BCR) sequences and 1,433,011 CD27+ sequences obtained by unbiased 5′RACE from 12 healthy controls. The 12 healthy controls comprised equal numbers of male and female and were made up of 4 Caucasian, 4 Asian, and 4 Hispanic individuals. The repertoire analysis demonstrated that less than 1% of the human repertoire comprises BCRs with CDR-H3s longer than 21 amino acids. A V-gene bias was observed in the long CDR3 subrepertoire, with IGHV1-69, IGHV4-34, IGHV1-18, and IGHV1-8 showing preferential enrichment in BCRs with long H3 loops. A bias against long loops was observed for IGHV3-23, IGHV4-59/61, IGHVS-51, IGHV3-48, IGHV3-53/66, IGHV3-15, IGHV3-74, IGHV3-73, IGHV3-72, and IGHV2-70. The IGHV4-34 scaffold was demonstrated to be autoreactive and had a short half-life.

Viable N-terminal and C-terminal CDR-H3 scaffold variation for long loops were also designed based on the 5′RACE reference repertoire. About 81,065 CDR-H3s of amino acid length 22 amino acids or greater were observed. By comparing across V-gene scaffolds, scaffold-specific H₃ stem variation was avoided as to allow the scaffold diversity to be cloned into multiple scaffold references.

Heterodimer Analysis

Heterodimer analysis was performed on scaffolds and variant sequences and lengths of the scaffolds were assayed.

Structural Analysis

Structural analysis was performed using GPCR scaffolds of variant sequences and lengths were assayed.

Example 6: Generation of GPCR Antibody Libraries

Based on GPCR-ligand interaction surfaces and scaffold arrangements, libraries were designed and de novo synthesized. See Example 4. 10 variant sequences were designed for the variable domain, heavy chain, 237 variant sequences were designed for the heavy chain complementarity determining region 3, and 44 variant sequences were designed for the variable domain, light chain. The fragments were synthesized as three fragments following similar methods as described in Examples 1-3.

Following de novo synthesis, 10 variant sequences were generated for the variable domain, heavy chain, 236 variant sequences were generated for the heavy chain complementarity determining region 3, and 43 variant sequences were designed for a region comprising the variable domain, light chain and CDR-L3 and of which 9 variants for variable domain, light chain were designed. This resulted in a library with about 10⁵ diversity (10×236×43). This was confirmed using next generation sequencing (NGS) with 16 million reads. The normalized sequencing reads for each of the 10 variants for the variable domain, heavy chain was about 1 (data not shown). The normalized sequencing reads for each of the 43 variants for the variable domain, light chain was about 1 (data not shown). The normalized sequencing reads for 236 variant sequences for the heavy chain complementarily determining region 3 were about 1 (data not shown).

The various light and heavy chains were then tested for expression and protein folding. The 10 variant sequences for variable domain, heavy chain included the following: IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, and IGHV4-59/61. Of the 10 variant sequences, IGHV1-18, IGHV1-69, and IGHV3-30/33rn exhibited improved characteristics such as improved thermostability. 9 variant sequences for variable domain, light chain included the following: IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, and IGLV2-14. Of the 9 variant sequences, IGKV1-39, IGKV3-15, IGLV1-51, and IGLV2-14 exhibited improved characteristics such as improved thermostability.

Example 7: Expression of GPCR Antibody Libraries in HEK293 Cells

Following generation of GPCR antibody libraries, about 47 GPCRs were selected for screening. GPCR constructs about 1.8 kb to about 4.5 kb in size were designed in a pCDNA3.1 vector. The GPCR constructs were then synthesized following similar methods as described in Examples 2-4 including hierarchal assembly. Of the 47 GPCR constructs, 46 GPCR constructs were synthesized.

The synthesized GPCR constructs were transfected in HEK293 and assayed for expression using immunofluorescence. HEK293 cells were transfected with the GPCR constructs comprising an N-terminally hemagglutinin (HA)-tagged human Y₁ receptor. Following 24-48 hours of transfection, cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde. Cells were stained using fluorescent primary antibody directed towards the HA tag or secondary antibodies comprising a fluorophore and DAPI to visualize the nuclei in blue. Human Y₁ receptor was visualized on the cell surface in non-permeabilized cells and on the cell surface and intracellularly in permeabilized cells.

GPCR constructs were also visualized by designing GPCR constructs comprising auto-fluorescent proteins. Human Y₁ receptor comprised EYFP fused to its C-terminus, and human Y₅ receptor comprised ECFP fused to its C-terminus. HEK293 cells were transfected with human Y₁ receptor or co-transfected with human Y₁ receptor and human Y₅ receptor. Following transfection cells were washed and fixed with 4% paraformaldehyde. Cells were stained with DAPI. Localization of human Y₁ receptor and human Y₅ receptor were visualized by fluorescence microscopy.

Example 8: Design of Immunoglobulin Library

An immunoglobulin scaffold library was designed for placement of GPCR binding domains and for improving stability for a range of GPCR binding domain encoding sequences. The immunoglobulin scaffold included a VH domain attached with a VL domain with a linker. Variant nucleic acid sequences were generated for the framework elements and CDR elements of the VH domain and VL domain. The structure of the design is shown in FIG. 8A. A full domain architecture is shown in FIG. 8B. Sequences for the leader, linker, and pIII are listed in Table 7.

TABLE 7 Nucleotide sequences SEQ ID NO Domain Sequence 16 Leader GCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAG CCGGCCATGGCC 17 Linker GCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGC TCTGGCGGTGGCGGATCGCATGCATCC 18 pIII CGCGCGGCCGCTGGAAGCGGCTCCCACCATCAC CATCACCAT

The VL domains that were designed include IGKV1-39, IGKV3-15, IGLV1-51, and IGLV2-14. Each of four VL domains were assembled with their respective invariant four framework elements (FW1, FW2, FW3, FW4) and variable 3 CDR (L1, L2, L3) elements. For IGKV1-39, there was 490 variants designed for L1, 420 variants designed for L2, and 824 variants designed for L3 resulting in a diversity of 1.7×10⁸ (490*420*824). For IGKV3-15, there was 490 variants designed for L1, 265 variants designed for L2, and 907 variants designed for L3 resulting in a diversity of 1.2×10⁸ (490*265*907). For IGLV 1-51, there was 184 variants designed for L1, 151 variants designed for L2, and 824 variants designed for L3 resulting in a diversity of 2.3×10⁷ (184*151*824). IGLV2-14, 967 variants designed for L1, 535 variants designed for L2, and 922 variants designed for L3 resulting in a diversity of 4.8 10⁸ (967*535*922). Table 8 lists the amino acid sequences and nucleotide sequences for the four framework elements (FW1, FW2, FW3, FW4) for IGLV 1-51. Table 9 lists the variable 3 CDR (L1, L2, L3) elements for IGLV 1-51. Variant amino acid sequences and nucleotide sequences for the four framework elements (FW1, FW2, FW3, FW4) and the variable 3 CDR (L1, L2, L3) elements were also designed for IGKV1-39, IGKV3-15, and IGLV2-14.

TABLE 8 Sequences for IGLV1-51 framework elements SEQ SEQ ID Amino Acid ID Element NO Sequence NO Nucleotide Sequence IGLV1-51 FW1 19 QSVLTQPPSVS 20 CAGTCTGTGTTGACGCAGCCG AAPGQKVTISC CCCTCAGTGTCTGCGGCCCCA GGACAGAAGGTCACCATCTCC TGC FW2 21 WYQQLPGTAPK 22 TGGTATCAGCAGCTCCCAGGA LLIY ACAGCCCCCAAACTCCTCATT TAT FW3 23 GIPDRFSGSKS 24 GGGATTCCTGACCGATTCTCT GGCTCCAAGTCTGGCACGTCA GTSATLGITGL GCCACCCTGGGCATCACCGGA QTGDEADYY CTCCAGACTGGGGACGAGGCC GATTATTAC FW4 25 GGGTKLTVL 26 GGCGGAGGGACCAAGCTGACC GTCCTA

TABLE 9 Sequences for IGLV1-51 CDR elements SEQ SEQ ID Amino Acid ID NO Sequence NO Nucleotide Sequence IGLV1-51-L1   27 SGSSSNIGSNHVS  210 TCTGGAAGCAGCTCCAACATTGGGAGTAATCATGTATCC   28 SGSSSNIGNNYLS  211 TCTGGAAGCAGCTCCAACATTGGGAATAATTATCTATCC   29 SGSSSNIANNYVS  212 TCTGGAAGCAGCTCCAACATTGCGAATAATTATGTATCC   30 SGSSPNIGNNYVS  213 TCTGGAAGCAGCCCCAACATTGGGAATAATTATGTATCG   31 SGSRSNIGSNYVS  214 TCTGGAAGCAGATCCAATATTGGGAGTAATTATGTTTCG   32 SGSSSNVGDNYVS  215 TCTGGAAGCAGCTCCAACGTTGGCGATAATTATGTTTCC   33 SGSSSNIGIQYVS  216 TCTGGAAGCAGCTCCAACATTGGGATTCAATATGTATCC   34 SGSSSNVGNNFVS  217 TCTGGAAGCAGCTCCAATGTTGGTAACAATTTTGTCTCC   35 SGSASNIGNNYVS  218 TCTGGAAGCGCCTCCAACATTGGGAATAATTATGTATCC   36 SGSGSNIGNNDVS  219 TCTGGAAGCGGCTCCAATATTGGGAATAATGATGTGTCC   37 SGSISNIGNNYVS  220 TCTGGAAGCATCTCCAACATTGGTAATAATTATGTATCC   38 SGSISNIGKNYVS  221 TCTGGAAGCATCTCCAACATTGGGAAAAATTATGTGTCG   39 SGSSSNIGHNYVS  222 TCTGGAAGCAGCTCCAACATTGGGCATAATTATGTATCG   40 PGSSSNIGNNYVS  223 CCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC   41 SGSTSNIGIHYVS  224 TCTGGAAGCACCTCCAACATTGGAATTCATTATGTATCC   42 SGSSSNIGSHYVS  225 TCTGGAAGCAGCTCCAACATTGGCAGTCATTATGTTTCC   43 SGSSSNIGNEYVS  226 TCCGGAAGCAGCTCCAACATTGGAAATGAATATGTATCC   44 SGSTSNIGNNYIS  227 TCTGGAAGCACCTCCAACATTGGAAATAATTATATATCG   45 SGSSSNIGNHFVS  228 TCTGGAAGCAGCTCCAATATTGGGAATCATTTTGTATCG   46 SGSSSNIGNNYVA  229 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTGGCC   47 SGSSSNIGSYYVS  230 TCTGGAAGCAGCTCCAACATTGGAAGTTATTATGTATCC   48 SGSGFNIGNNYVS  231 TCTGGAAGTGGTTTCAACATTGGGAATAATTATGTCTCT   49 SGSTSNIGNNYVS  232 TCTGGAAGCACCTCCAACATTGGGAATAATTATGTGTCC   50 SGSSSDIGNNYVS  233 TCTGGAAGCAGCTCCGACATTGGCAATAATTATGTATCC   51 SGSSSNIGNNVVS  234 TCTGGAAGCAGCTCCAACATTGGGAATAATGTTGTATCC   52 SGSKSNIGKNYVS  235 TCTGGAAGCAAGTCTAACATTGGGAAAAATTATGTATCC   53 SGSSTNIGNNYVS  236 TCTGGAAGCAGCACCAACATTGGGAATAATTATGTATCC   54 SGSISNIGDNYVS  237 TCTGGAAGCATCTCCAACATTGGGGATAATTATGTATCC   55 SGSSSNIGSKDVS  238 TCTGGAAGCAGCTCCAACATTGGGAGTAAGGATGTATCA   56 SGSSSNIENNDVS  239 TCTGGAAGCAGCTCCAACATTGAGAATAATGATGTATCG   57 SGSSSNIGNHYVS  240 TCTGGAAGCAGCTCCAACATTGGGAATCATTATGTATCC   58 SGSSSNIGKDFVS  241 TCTGGAAGCAGCTCCAACATTGGGAAGGATTTTGTCTCC   59 SGSTSNIGSNFVS  242 TCTGGCAGTACTTCCAACATCGGAAGTAATTTTGTTTCC   60 SGSTSNIGHNYVS  243 TCTGGAAGCACCTCCAACATTGGGCATAATTATGTATCC   61 SASSSNIGNNYVS  244 TCTGCAAGCAGCTCCAACATTGGGAATAATTATGTATCC   62 SGSSSSIGNNYVS  245 TCTGGAAGCAGCTCCAGCATTGGCAATAATTATGTATCC   63 SGSSSTIGNNYVS  246 TCTGGAAGCAGCTCCACCATTGGGAATAATTATGTATCC   64 SGSSSNIENNYVS  247 TCTGGAAGCAGCTCCAACATTGAAAATAATTATGTATCC   65 SGSSSNIGNQYVS  248 TCTGGAAGCAGCTCCAACATTGGGAATCAGTATGTATCC   66 SGSSSNIGNNYVF  249 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATTC   67 SGSSSNIGRNYVS  250 TCTGGAAGCAGCTCCAACATTGGGAGGAATTATGTCTCC   68 SGGSSNIGNYYVS  251 TCTGGAGGCAGCTCCAACATTGGAAATTATTATGTATCG   69 SGSSSNIGDNYVS  252 TCTGGAAGCAGCTCCAACATTGGAGATAATTATGTCTCC   70 SGGSSNIGINYVS  253 TCTGGAGGCAGCTCCAACATTGGAATTAATTATGTATCC   71 SGGSSNIGKNYVS  254 TCTGGAGGCAGCTCCAACATTGGGAAGAATTATGTATCC   72 SGSSSNIGKRSVS  255 TCTGGAAGCAGCTCCAACATTGGGAAGAGATCTGTATCG   73 SGSRSNIGNNYVS  256 TCTGGAAGCAGATCCAACATTGGGAATAACTATGTATCC   74 SGSSSNIGNNLVS  257 TCGGGAAGCAGCTCCAACATTGGGAATAATCTTGTTTCC   75 SGSSSNIGINYVS  258 TCTGGAAGCAGCTCCAACATTGGGATCAATTATGTATCC   76 SGSSSNIGNNFVS  259 TCTGGAAGCAGCTCCAACATCGGGAATAATTTTGTATCC   77 SGTSSNIGRNFVS  260 TCTGGAACCAGCTCCAACATTGGCAGAAATTTTGTATCC   78 SGRRSNIGNNYVS  261 TCTGGAAGGAGGTCCAACATTGGAAATAATTATGTGTCC   79 SGGSFNIGNNYVS  262 TCTGGAGGCAGCTTCAATATTGGGAATAATTATGTATCC   80 SGSTSNIGENYVS  263 TCTGGAAGCACTTCCAACATTGGGGAGAATTATGTGTCC   81 SGSSSNIGSDYVS  264 TCTGGAAGCAGCTCCAATATTGGGAGTGATTATGTATCC   82 SGTSSNIGSNYVS  265 TCTGGAACCAGCTCCAACATTGGGAGTAATTATGTATCC   83 SGSSSNIGTNFVS  266 TCTGGAAGCAGCTCCAACATTGGGACTAATTTTGTATCC   84 SGSSSNFGNNYVS  267 TCTGGAAGCAGCTCCAACTTTGGGAATAATTATGTATCC   85 SGSTSNIGNNHVS  268 TCTGGAAGCACCTCCAACATTGGGAATAATCATGTATCC   86 SGSSSNIGNDFVS  269 TCTGGAAGCAGCTCCAACATTGGGAATGATTTTGTATCC   87 SGSSSDIGDNYVS  270 TCTGGAAGCAGCTCCGACATTGGCGATAATTATGTGTCC   88 SGSSSNIGKYYVS  271 TCTGGAAGCAGCTCCAACATTGGGAAATATTATGTATCC   89 SGSSSNIGGNYVS  272 TCTGGAAGCAGCTCCAACATTGGCGGTAATTATGTATCC   90 SGSSSNTGNNYVS  273 TCTGGAAGCAGCTCCAACACTGGGAATAATTATGTATCC   91 SGSSSNVGNNYVS  274 TCTGGAAGCAGCTCCAACGTTGGGAATAATTATGTGTCT   92 SGSSSNIANNFVS  275 TCTGGAAGCAGCTCCAACATTGCGAATAATTTTGTATCC   93 SGSSSNIGNDYVS  276 TCTGGAAGCAGCTCCAACATTGGGAATGATTATGTATCC   94 SGSTSNIENNYVS  277 TCTGGAAGCACCTCCAATATTGAGAATAATTATGTTTCC   95 SGGSSNIGNNDVS  278 TCTGGAGGCAGCTCCAATATTGGCAATAATGATGTGTCC   96 SGSTSNIGNHYVS  279 TCTGGAAGCACCTCCAACATTGGGAATCATTATGTATCC   97 SGSSSNIGDNDVS  280 TCAGGAAGCAGCTCCAATATTGGGGATAATGATGTATCC   98 SGYSSNIGNNYVS  281 TCTGGATACAGCTCCAACATTGGGAATAATTATGTATCC   99 SGSGSNIGNNFVS  282 TCTGGAAGCGGCTCCAACATTGGAAATAATTTTGTATCC  100 SGSSSNIWNNYVS  283 TCTGGAAGCAGCTCCAACATTTGGAATAATTATGTATCC  101 FGSSSNIGNNYVS  284 TTTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC  102 SGSSSNIEKNYVS  285 TCTGGAAGCAGCTCCAACATTGAGAAGAATTATGTATCC  103 SGSRSNIGNYYVS  286 TCTGGAAGTAGATCCAATATTGGAAATTATTATGTATCC  104 SGTKSNIGNNYVS  287 TCTGGAACCAAGTCAAACATTGGGAATAATTATGTATCT  105 SGSTSNIGNYYVS  288 TCTGGAAGCACCTCCAACATTGGGAATTATTATGTATCC  106 SGTSSNIGNNYVA  289 TCTGGAACCAGCTCCAACATTGGGAATAATTATGTGGCC  107 PGTSSNIGNNYVS  290 CCTGGAACCAGCTCCAACATTGGGAATAATTATGTATCC  108 SGSTSNIGINYVS  291 TCCGGAAGCACCTCCAACATTGGGATTAATTATGTATCC  109 SGSSSNIGSNLVS  292 TCTGGAAGCAGCTCCAACATTGGGAGTAATCTGGTATCC  110 SGSSSNIENNHVS  293 TCTGGAAGCAGCTCCAACATTGAGAATAATCATGTATCC  111 SGTRSNIGNNYVS  294 TCTGGAACCAGGTCCAACATCGGCAATAATTATGTTTCG  112 SGSTSNIGDNYVS  295 TCTGGAAGCACCTCCAACATTGGGGACAATTATGTTTCC  113 SGGSSNIGKNFVS  296 TCTGGAGGCAGTTCCAACATTGGGAAGAATTTTGTATCC  114 SGSRSDIGNNYVS  297 TCTGGAAGCAGGTCCGACATTGGGAATAATTATGTATCC  115 SGTSSNIGNNDVS  298 TCTGGAACTAGCTCCAACATTGGGAATAATGATGTATCC  116 SGSSSNIGSKYVS  299 TCTGGAAGCAGCTCCAACATTGGGAGTAAATATGTATCA  117 SGSSFNIGNNYVS  300 TCTGGAAGCAGCTTCAACATTGGGAATAATTATGTATCC  118 SGSSSNIGNTYVS  301 TCTGGAAGCAGCTCCAACATTGGGAATACTTATGTATCC  119 SGSSSNIGDNHVS  302 TCTGGAAGCAGCTCCAATATTGGGGATAATCATGTATCC  120 SGSSSNIGNNHVS  303 TCTGGAAGCAGCTCCAACATTGGCAATAATCATGTTTCC  121 SGSTSNIGNNDVS  304 TCTGGAAGCACCTCCAACATTGGGAATAATGATGTATCC  122 SGSRSNVGNNYVS  305 TCTGGAAGCAGATCCAACGTTGGCAATAATTATGTTTCA  123 SGGTSNIGKNYVS  306 TCCGGAGGCACCTCCAACATTGGGAAGAATTATGTGTCT  124 SGSSSNIADNYVS  307 TCTGGAAGCAGCTCCAACATTGCCGATAATTATGTTTCC  125 SGSSSNIGANYVS  308 TCTGGAAGCAGCTCCAACATTGGCGCCAATTATGTATCC  126 SGSSSNIGSNYVA  309 TCTGGAAGCAGCTCCAACATTGGGAGTAATTATGTGGCC  127 SGSSSNIGNNFLS  310 TCTGGAAGCAGCTCCAACATTGGGAACAATTTTCTCTCC  128 SGRSSNIGKNYVS  311 TCTGGAAGAAGCTCCAACATTGGGAAGAATTATGTATCC  129 SGSSPNIGANYVS  312 TCTGGAAGCAGCCCCAACATTGGGGCTAATTATGTATCC  130 SGSSSNIGPNYVS  313 TCCGGAAGCAGCTCCAACATTGGGCCTAATTATGTGTCC  131 SGSSSTIGNNYIS  314 TCTGGAAGCAGCTCCACCATTGGGAATAATTATATATCC  132 SGSSSNIGNYFVS  315 TCTGGAAGCAGCTCCAACATTGGGAATTATTTTGTATCC  133 SGSRSNIGNNFVS  316 TCTGGAAGCCGCTCCAACATTGGTAATAATTTTGTATCC  134 SGGSSNIGSNFVS  317 TCTGGAGGCAGCTCCAACATTGGGAGTAATTTTGTATCC  135 SGSSSNIGYNYVS  318 TCTGGAAGCAGCTCCAACATTGGGTATAATTATGTATCC  136 SGTSSNIENNYVS  319 TCTGGAACCAGCTCGAACATTGAGAACAATTATGTATCC  137 SGSSSNIGNYYVS  320 TCTGGAAGTAGCTCCAACATTGGGAATTATTATGTATCC  138 SGSTSNIGKNYVS  321 TCTGGAAGCACCTCCAACATTGGGAAGAATTATGTATCC  139 SGSSSNIGTYYVS  322 TCTGGAAGCAGTTCCAACATTGGGACTTATTATGTCTCT  140 SGSSSNVGKNYVS  323 TCTGGAAGCAGCTCCAACGTTGGGAAAAATTATGTATCT  141 SGSTSNIGDNFVS  324 TCTGGAAGCACCTCCAACATTGGGGATAATTTTGTATCC  142 SGSTSNIGTNYVS  325 TCTGGAAGCACCTCCAACATTGGAACTAATTATGTTTCC  143 SGGTSNIGNNYVS  326 TCTGGAGGTACTTCCAACATTGGGAATAATTATGTCTCC  144 SGSYSNIGNNYVS  327 TCTGGAAGCTACTCCAATATTGGGAATAATTATGTATCC  145 SGSSSNIEDNYVS  328 TCTGGAAGCAGCTCCAACATTGAAGATAATTATGTATCC  146 SGSSSNIGKHYVS  329 TCTGGAAGCAGCTCCAACATTGGGAAACATTATGTATCC  147 SGSGSNIGSNYVS  330 TCCGGTTCCGGCTCAAACATTGGAAGTAATTATGTCTCC  148 SGSSSNIGNNYIS  331 TCTGGAAGCAGCTCCAACATTGGAAATAATTATATATCA  149 SGASSNIGNNYVS  332 TCTGGAGCCAGTTCCAACATTGGGAATAATTATGTTTCC  150 SGRTSNIGNNYVS  333 TCTGGACGCACCTCCAACATCGGGAACAATTATGTATCC  151 SGGSSNIGSNYVS  334 TCTGGAGGCAGCTCCAATATTGGGAGTAATTACGTATCC  152 SGSGSNIGNNYVS  335 TCTGGAAGCGGCTCCAACATTGGGAATAATTATGTATCC  153 SGSTSNIGSNYVS  336 TCTGGAAGCACCTCCAACATTGGGAGTAATTATGTATCC  154 SGSSSSIGNNYVA  337 TCTGGAAGCAGCTCCAGCATTGGGAATAATTATGTGGCG  155 SGSSSNLGNNYVS  338 TCTGGAAGCAGTTCCAACCTTGGAAATAATTATGTATCC  156 SGTSSNIGKNYVS  339 TCTGGAACCAGCTCCAACATTGGGAAAAATTATGTATCC  157 SGSSSDIGNKYIS  340 TCTGGAAGCAGCTCCGATATTGGGAACAAGTATATATCC  158 SGSSSNIGSNYIS  341 TCTGGAAGCAGCTCCAACATTGGAAGTAATTACATATCC  159 SGSTSNIGANYVS  342 TCTGGAAGCACCTCCAACATTGGGGCTAACTATGTGTCC  160 SGSSSNIGNKYVS  343 TCTGGAAGCAGCTCCAACATTGGGAATAAGTATGTATCC  161 SGSSSNIGNNYGS  344 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGGATCC  162 SGSTSNIANNYVS  345 TCTGGAAGCACCTCCAACATTGCGAATAATTATGTATCC  163 SGSYSNIGSNYVS  346 TCTGGAAGCTACTCCAATATTGGGAGTAATTATGTATCC  164 SGSSSNIGSNFVS  347 TCTGGAAGCAGCTCCAACATTGGGAGTAATTTTGTATCC  165 SGSSSNLENNYVS  348 TCTGGAAGCAGCTCCAATCTTGAGAATAATTATGTATCC  166 SGSISNIGSNYVS  349 TCTGGAAGCATCTCCAATATTGGCAGTAATTATGTATCC  167 SGSSSDIGSNYVS  350 TCTGGAAGCAGCTCCGACATTGGGAGTAATTATGTATCC  168 SGSSSNIGTNYVS  351 TCTGGAAGCAGCTCCAACATTGGGACTAATTATGTATCC  169 SGSSSNIGKNFVS  352 TCTGGAAGCAGCTCCAACATTGGGAAGAATTTTGTATCC  170 SGSSSNIGNNFIS  353 TCTGGAAGCAGCTCCAACATTGGGAATAATTTTATATCC  171 SGGSSNIGNNYVS  354 TCTGGAGGCAGCTCCAACATTGGCAATAATTATGTTTCC  172 SGSSSNIGENYVS  355 TCTGGAAGCAGCTCCAACATTGGGGAGAATTATGTATCC  173 SGSSSNIGNNFVA  356 TCTGGAAGCAGCTCCAATATTGGGAATAATTTTGTGGCC  174 SGGSSNIGNNYVA  357 TCTGGAGGCAGCTCCAACATTGGGAATAATTATGTAGCC  175 SGSSSHIGNNYVS  358 TCTGGAAGCAGCTCCCACATTGGAAATAATTATGTATCC  176 SGSSSNIGSNDVS  359 TCTGGAAGCAGCTCCAATATTGGAAGTAATGATGTATCG  177 SGSSSNIGNNYVT  360 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTAACC  178 SGSSSNIGNNPVS  361 TCTGGAAGCAGCTCCAACATTGGGAATAATCCTGTATCC  179 SGGSSNIGNHYVS  362 TCTGGAGGCAGCTCCAATATTGGGAATCATTATGTATCC  180 SGTSSNIGNNYVS  363 TCTGGAACCAGCTCCAACATTGGGAATAATTATGTATCC  181 SGSSSNIGSNYVS  364 TCTGGAAGCAGCTCCAACATTGGAAGTAATTATGTCTCG  182 SGGTSNIGSNYVS  365 TCTGGAGGCACCTCCAACATTGGAAGTAATTATGTATCC  183 SGSKSNIGNNYVS  366 TCTGGAAGCAAGTCCAACATTGGGAATAATTATGTATCC  184 SGRSSNIGNNYVS  367 TCTGGAAGAAGCTCCAACATTGGGAATAATTATGTATCG  185 SGSSSNVGSNYVS  368 TCTGGAAGCAGCTCCAACGTTGGGAGTAATTATGTTTCC  186 SGSTSNIGNNFVS  369 TCTGGAAGCACCTCCAATATTGGGAATAATTTTGTATCC  187 SGSNFNIGNNYVS  370 TCTGGAAGCAACTTCAACATTGGGAATAATTATGTCTCC  188 SGSTSNIGYNYVS  371 TCTGGAAGCACCTCCAATATTGGATATAATTATGTATCC  189 SGSSSNIVSNYVS  372 TCTGGAAGCAGCTCCAATATTGTAAGTAATTATGTATCC  190 SGTSSNIGNNFVS  373 TCTGGAACCAGCTCCAACATTGGGAATAATTTTGTATCC  191 SGSSSNIGRNFVS  374 TCTGGAAGCAGCTCCAACATTGGGAGGAATTTTGTGTCC  192 SGTTSNIGNNYVS  375 TCTGGAACGACCTCCAACATTGGGAATAATTATGTCTCC  193 SGSSSNIGNNDVS  376 TCTGGAAGCAGCTCCAACATTGGGAATAATGATGTATCC  194 SGSSSNIGNHDVS  377 TCTGGAAGCAGCTCCAACATTGGGAATCATGATGTATCC  195 SGSSSNIGSSHVS  378 TCTGGAAGCAGCTCCAACATTGGAAGTAGTCATGTATCC  196 SGSSSNIGIHYVS  379 TCTGGAAGCAGCTCCAACATTGGGATTCATTATGTATCC  197 SGGGSNIGYNYVS  380 TCTGGAGGCGGCTCCAACATTGGCTATAATTATGTCTCC  198 SGSSSNIGDHYVS  381 TCTGGAAGCAGCTCCAACATTGGGGATCATTATGTGTCG  199 SGSSSNLGKNYVS  382 TCTGGAAGCAGCTCCAACCTTGGGAAGAATTATGTATCT  200 SGSSSNIGDNFVS  383 TCTGGAAGCAGCTCCAACATTGGCGATAATTTTGTATCC  201 SGSTSNIEKNYVS  384 TCTGGAAGCACCTCCAACATTGAGAAAAACTATGTATCG  202 SGSSSNIGKDYVS  385 TCTGGAAGCAGCTCCAACATTGGGAAGGATTATGTATCC  203 SGSSSNIGKNYVS  386 TCTGGAAGCAGCTCCAACATTGGGAAGAATTATGTATCC  204 SGSSSNIGNNYVS  387 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC  205 SGSSSNIGNNYAS  388 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGCCTCC  206 SGISSNIGNNYVS  389 TCTGGAATCAGCTCCAACATTGGGAATAATTATGTATCC  207 TGSSSNIGNNYVS  390 ACTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCC  208 SGTSSNIGNNHVS  391 TCTGGAACCAGCTCCAACATTGGGAATAATCATGTTTCC  209 SGSRSNIGKNYVS  392 TCTGGAAGTCGTTCCAACATTGGGAAAAATTATGTATCC IGLV1-51-L2  393 DNNKRPP  544 GACAATAATAAGCGACCCCCA  394 ENNRRPS  545 GAGAATAATAGGCGACCCTCA  395 DNNKQPS  546 GACAATAATAAGCAACCCTCA  396 DNNKRPL  547 GACAATAACAAGCGACCCTTG  397 DNDKRPA  548 GACAATGATAAGCGACCCGCA  398 DNHERPS  549 GACAATCATGAGCGACCCTCA  399 ENRKRPS  550 GAAAACCGTAAGCGACCCTCA  400 DNDQRPS  551 GACAATGATCAGCGACCCTCA  401 ENYKRPS  552 GAGAATTATAAGCGACCCTCA  402 ENTKRPS  553 GAAAATACTAAGCGACCCTCA  403 DTEKRPS  554 GACACTGAGAAGAGGCCCTCA  404 DNDKRPP  555 GACAATGATAAGCGACCCCCA  405 DHNKRPS  556 GACCATAATAAGCGACCCTCA  406 GNNERPS  557 GGCAATAATGAGCGACCCTCA  407 DTSKRPS  558 GACACTAGTAAGCGACCCTCA  408 EYNKRPS  559 GAATATAATAAGCGCCCCTCA  409 ENIKRPS  560 GAAAATATTAAGCGACCCTCA  410 DNVKRPS  561 GACAATGTTAAGCGACCCTCA  411 ENDKRSS  562 GAAAACGATAAACGATCCTCA  412 ENNKRHS  563 GAAAATAATAAGCGACACTCA  413 GNDQRPS  564 GGAAATGATCAGCGACCCTCA  414 DNDRRPS  565 GACAATGATAGGCGACCCTCA  415 DNHKRPS  566 GACAATCATAAGCGGCCCTCA  416 DNNDRPS  567 GACAATAATGACCGACCCTCA  417 ENNQRPS  568 GAGAATAATCAGCGACCCTCA  418 DNNQRPS  569 GACAATAATCAGCGACCCTCA  419 ENVKRPS  570 GAGAATGTTAAGCGACCCTCA  420 DTYKRPS  571 GACACTTATAAGAGACCCTCA  421 NNNNRPS  572 AACAATAATAACCGACCCTCA  422 GNNNRPS  573 GGCAATAATAATCGACCCTCA  423 ENDQRPS  574 GAAAATGATCAGCGACCCTCA  424 DNNKRAS  575 GACAATAATAAGCGAGCCTCA  425 DNDKRPL  576 GACAATGATAAGCGACCCTTA  426 DTDERPS  577 GACACTGATGAGCGACCTTCA  427 DNRKRPS  578 GACAATAGGAAGCGACCCTCA  428 DNDARPS  579 GACAATGATGCTCGACCCTCA  429 DNNKRLS  580 GACAATAATAAGCGACTCTCA  430 DNDKRAS  581 GACAATGATAAGCGAGCCTCA  431 DNTERPS  582 GACAATACTGAGCGACCCTCA  432 DNNIRPS  583 GACAATAATATTCGACCCTCA  433 DNKRRPS  584 GACAATAAGAGGCGACCCTCA  434 DDNNRPS  585 GACGATAATAACCGACCCTCA  435 ANNRRPS  586 GCGAATAATCGACGACCCTCA  436 DNDKRLS  587 GACAATGATAAGCGACTGTCA  437 DNNKRPA  588 GACAATAATAAGCGACCCGCA  438 DNYRRPS  589 GACAATTATAGACGTCCCTCA  439 ANDQRPS  590 GCCAATGATCAGCGACCCTCA  440 DNDKRRS  591 GACAATGATAAGCGACGCTCA  441 DKNERPS  592 GACAAGAATGAGCGACCCTCA  442 DNKERPS  593 GACAATAAGGAGCGACCCTCA  443 DNNKGPS  594 GACAATAATAAGGGACCCTCA  444 ENDRRPS  595 GAAAATGATAGACGACCCTCA  445 ENDERPS  596 GAAAATGATGAGCGACCCTCA  446 QNNKRPS  597 CAAAATAATAAGCGACCCTCA  447 DNRERPS  598 GACAATCGTGAGCGACCCTCA  448 DNNRRPS  599 GACAATAATAGACGACCCTCA  449 GNNRRPS  600 GGAAATAATAGGCGACCCTCA  450 DNDNRPS  601 GACAATGATAACCGACCCTCA  451 EDNKRPS  602 GAAGATAATAAGCGACCCTCA  452 DDDERPS  603 GACGATGATGAGCGGCCCTCA  453 ASNKRPS  604 GCAAGTAATAAGCGACCCTCA  454 DNNKRSS  605 GACAATAATAAGCGATCCTCA  455 QNNERPS  606 CAAAATAATGAGCGACCCTCA  456 DDDRRPS  607 GACGATGATAGGCGACCCTCA  457 NNDKRPS  608 AACAATGATAAGCGACCCTCA  458 DNNNRPS  609 GACAATAATAACCGACCCTCA  459 DNNVRPS  610 GACAATAATGTGCGACCCTCA  460 ENNERPS  611 GAAAATAATGAGCGACCCTCA  461 DNNHRPS  612 GACAATAATCACCGACCCTCA  462 DNDERPS  613 GACAATGATGAGCGCCCCTCG  463 DNIRRPS  614 GACAATATCCGGCGACCCTCA  464 DFNKRPS  615 GACTTTAATAAGCGACCCTCA  465 ETNKRPS  616 GAAACTAATAAGCGACCCTCA  466 NDNKRPS  617 AACGATAATAAGCGACCCTCA  467 DDNKRPS  618 GACGATAATAAGCGACCCTCA  468 DNYKRPS  619 GACAATTATAAGCGACCCTCA  469 HNNKRPS  620 CACAATAATAAGCGACCCTCA  470 DNHQRPS  621 GACAATCATCAGCGACCCTCA  471 DNYKRAS  622 GACAATTATAAGCGAGCCTCA  472 DNIKRPS  623 GACAATATTAAGCGACCCTCA  473 DTHKRPS  624 GACACTCATAAGCGACCCTCA  474 DTNRRPS  625 GACACTAATAGGCGACCCTCT  475 DTNQRPS  626 GACACTAATCAGCGACCCTCA  476 ESDKRPS  627 GAAAGTGATAAGCGACCCTCA  477 DNDKRSS  628 GACAATGATAAGCGATCTTCG  478 GSNKRPS  629 GGCAGTAATAAGCGACCCTCA  479 DNNKRVS  630 GACAATAACAAGCGAGTTTCA  480 NNNRRPS  631 AACAATAATAGGCGACCCTCA  481 DNFKRPS  632 GACAATTTTAAGCGACCCTCA  482 ENDKRPS  633 GAAAATGATAAACGACCCTCA  483 ENNKRLS  634 GAAAATAATAAGCGACTCTCA  484 ADNKRPS  635 GCAGATAATAAGCGACCCTCA  485 EDNERPS  636 GAAGATAATGAGCGCCCCTCA  486 DTDQRPS  637 GACACTGATCAGCGACCCTCA  487 DNYQRPS  638 GACAATTATCAGCGACCCTCA  488 DENKRPS  639 GACGAGAATAAGCGACCCTCA  489 DTNKRPS  640 GACACTAATAAGCGACCCTCA  490 DDYRRPS  641 GACGATTATCGGCGACCCTCA  491 DNDKRHS  642 GACAACGATAAGCGGCACTCA  492 ENDNRPS  643 GAAAATGATAATCGACCCTCA  493 DDNERPS  644 GACGATAATGAGCGCCCCTCA  494 DNKKRPS  645 GACAATAAGAAGCGACCCTCA  495 DVDKRPS  646 GACGTTGATAAGCGACCCTCA  496 ENKKRPS  647 GAAAATAAAAAACGACCCTCT  497 VNDKRPS  648 GTCAATGATAAGCGACCCTCA  498 DNDHRPS  649 GACAATGATCACCGACCCTCA  499 DINKRPS  650 GACATTAATAAGCGACCCTCA  500 ANNERPS  651 GCCAATAATGAGCGACCCTCA  501 DNENRPS  652 GACAATGAAAACCGACCGTCA  502 GDDKRPS  653 GGCGATGATAAGCGACCCTCA  503 ANNQRPS  654 GCCAATAATCAGCGACCTTCA  504 DDDKRPS  655 GACGATGATAAGCGACCCTCA  505 YNNKRPS  656 TACAATAATAAGCGGCCCTCA  506 EDDKRPS  657 GAAGATGATAAGCGACCCTCA  507 ENNNRPS  658 GAAAACAATAACCGACCCTCG  508 DNNLRPS  659 GACAATAATCTGCGACCCTCA  509 ESNKRPS  660 GAGAGTAACAAGCGACCCTCA  510 DTDKRPS  661 GACACTGATAAGCGGCCCTCA  511 DDDQRPS  662 GACGATGATCAGCGACCCTCA  512 VNNKRPS  663 GTGAATAATAAGAGACCCTCC  513 DDYKRPS  664 GACGATTATAAGCGACCCTCA  514 DNTKRPS  665 GACAATACTAAGCGACCCTCA  515 DDTERPS  666 GACGATACTGAGCGACCCTCA  516 GNDKRPS  667 GGCAATGATAAGCGACCCTCA  517 DNEKRPS  668 GACAATGAAAAGCGACCCTCA  518 DNDDRPS  669 GACAATGATGACCGACCCTCA  519 DDNRRPS  670 GACGATAATAGGCGTCCCTCA  520 GNNKRPS  671 GGCAATAATAAGCGACCCTCA  521 ANDKRPS  672 GCCAATGATAAGCGACCCTCA  522 DNNKRHS  673 GACAATAATAAGCGACACTCA  523 DDNQRPS  674 GACGACAATCAGCGACCCTCA  524 GNDRRPS  675 GGCAATGATAGGCGACCCTCA  525 DNHNRPS  676 GACAATCATAACCGACCCTCA  526 DNYERPS  677 GACAATTATGAGCGACCCTCA  527 ENNKRSS  678 GAAAATAATAAGCGATCCTCA  528 DDHKRPS  679 GACGATCATAAGCGGCCCTCA  529 DNNKRRS  680 GACAATAATAAACGACGTTCA  530 DNDKRPS  681 GACAATGATAAGCGACCGTCA  531 DKNKRPS  682 GACAAGAATAAGCGACCCTCA  532 DNNKRPS  683 GACAATAATAAGCGACCCTCA  533 DIDKRPS  684 GACATTGATAAGCGACCCTCA  534 DDKKRPS  685 GACGATAAGAAGCGACCCTCA  535 ANNKRPS  686 GCCAATAATAAGCGACCCTCA  536 DNDKGPS  687 GACAATGATAAGGGACCCTCA  537 EDNRRPS  688 GAAGATAATAGGCGACCCTCA  538 ENNKRPS  689 GAGAATAATAAGCGACCCTCA  539 NNNKRPS  690 AACAATAATAAGCGACCCTCA  540 DNNERPS  691 GACAATAATGAGCGACCCTCA  541 DNIQRPS  692 GACAATATTCAGCGACCCTCA  542 DNNYRPS  693 GACAATAATTACCGACCCTCA  543 DNYNRPS  694 GACAATTATAACCGACCCTCA IGLV1-51-L3  695 CGTWDTSLSAVVF 1431 TGCGGAACATGGGATACCAGCCTGAGTGCTGTGGTGTTC  696 CGTWDTSLSAGVF 1432 TGCGGAACATGGGATACCAGCCTGAGTGCTGGGGTGTTC  697 CGTWDTSLSAWVF 1433 TGCGGAACATGGGATACCAGCCTGAGTGCTTGGGTGTTC  698 CGTWDRSLSAGVF 1434 TGCGGAACATGGGATAGGAGCCTGAGTGCGGGGGTGTTC  699 CGTWDRSLSAWVF 1435 TGCGGAACATGGGATAGGAGCCTGAGTGCTTGGGTATTT  700 CGTWDTSLSGGVF 1436 TGCGGAACATGGGATACCAGCCTGAGTGGTGGGGTGTTC  701 CGTWDTSLRAGVF 1437 TGCGGAACATGGGATACTAGCCTGCGTGCTGGCGTCTTC  702 CGTWDRSLSVWVF 1438 TGCGGAACATGGGATAGGAGCCTGAGTGTTTGGGTGTTC  703 CGTWDTSLSVVVF 1439 TGCGGAACATGGGATACCAGTCTGAGTGTTGTGGTCTTC  704 CGTWDTSLSAAVF 1440 TGCGGAACGTGGGATACCAGCCTGAGTGCTGCGGTGTTC  705 CGAWDTSLSAGVF 1441 TGCGGAGCATGGGATACCAGCCTGAGTGCTGGAGTGTTC  706 CATWDTSLSAVVF 1442 TGCGCAACATGGGATACCAGCCTGAGTGCTGTGGTATTC  707 CATWDTSLSAGVF 1443 TGCGCAACATGGGATACCAGCCTGAGTGCTGGTGTGTTC  708 CGTWESSLSAWVF 1444 TGTGGAACATGGGAGAGCAGCCTGAGTGCTTGGGTGTTC  709 CGTWDTTLSAGVF 1445 TGCGGAACATGGGATACCACCCTGAGTGCGGGTGTCTTC  710 CGTWDTSLSVWVF 1446 TGCGGAACATGGGATACTAGCCTGAGTGTGTGGGTGTTC  711 CGTWDTSLSVGVF 1447 TGCGGAACATGGGATACTAGCCTGAGTGTTGGGGTGTTC  712 CGTWDTSLSTGVF 1448 TGCGGAACATGGGACACCAGTCTGAGCACTGGCGTCTTC  713 CGTWDTSLSGVVF 1449 TGCGGAACATGGGATACCAGCCTGAGTGGTGTGGTCTTC  714 CGTWDTSLSAYVF 1450 TGCGGAACATGGGATACCAGCCTGAGTGCTTATGTCTTC  715 CGTWDTSLSAEVF 1451 TGCGGAACATGGGATACCAGCCTGAGTGCTGAGGTGTTC  716 CGTWDTGLSAGVF 1452 TGCGGAACATGGGATACCGGCCTGAGTGCTGGGGTATTC  717 CGTWDRSLSAYVF 1453 TGCGGAACGTGGGATAGGAGCCTGAGTGCTTATGTCTTC  718 CGTWDRSLSAVVF 1454 TGCGGAACATGGGATAGGAGCCTCAGTGCCGTGGTATTC  719 CGTWDNTLSAWVF 1455 TGCGGAACATGGGATAACACCCTGAGTGCGTGGGTGTTC  720 CGTWDNRLSAGVF 1456 TGCGGAACATGGGATAACAGGCTGAGTGCTGGGGTGTTC  721 CGTWDISLSAWVF 1457 TGCGGAACATGGGACATCAGCCTGAGTGCTTGGGTGTTC  722 CGTWHSSLSAGVF 1458 TGCGGAACATGGCATAGCAGCCTGAGTGCTGGGGTATTC  723 CGTWGSSLSAWVF 1459 TGCGGAACATGGGGTAGCAGTTTGAGTGCTTGGGTGTTC  724 CGTWESSLSGWVF 1460 TGCGGAACATGGGAGAGCAGCCTGAGTGGTTGGGTGTTC  725 CGTWESSLSAVVF 1461 TGCGGAACATGGGAGAGCAGCCTGAGTGCTGTGGTTTTC  726 CGTWDYSLSAVVF 1462 TGCGGAACATGGGATTACAGCCTGAGTGCTGTGGTATTC  727 CGTWDYSLSAGVF 1463 TGCGGAACATGGGATTACAGCCTGAGTGCTGGGGTATTC  728 CGTWDVSLSVGVF 1464 TGCGGAACATGGGATGTCAGCCTGAGTGTTGGAGTGTTC  729 CGTWDTTLSAVVF 1465 TGCGGAACATGGGATACCACCCTGAGTGCTGTGGTTTTC  730 CGTWDTTLNIGVF 1466 TGCGGAACATGGGATACCACTCTGAATATTGGGGTGTTC  731 CGTWDTSLTAVVF 1467 TGCGGAACATGGGATACCAGCCTGACTGCTGTGGTATTC  732 CGTWDTSLTAAVF 1468 TGCGGAACCTGGGATACCAGCCTGACTGCTGCTGTGTTC  733 CGTWDTSLSVGLF 1469 TGCGGCACATGGGATACCAGCCTGAGTGTGGGGCTATTC  734 CGTWDTSLSGRVF 1470 TGCGGAACCTGGGATACCAGCCTGAGTGGTAGGGTGTTC  735 CGTWDTSLSGAVF 1471 TGCGGAACATGGGATACCAGCCTGAGTGGTGCAGTGTTC  736 CGTWDTSLSAGLF 1472 TGCGGAACATGGGATACCAGCCTGAGTGCTGGCCTGTTC  737 CGTWDTSLSAGGVF 1473 TGCGGAACATGGGATACCAGCCTGAGTGCTGGAGGGGTCTTC  738 CGTWDTSLRAYVF 1474 TGCGGAACATGGGATACCAGCCTGCGTGCTTATGTCTTC  739 CGTWDTSLRAWVF 1475 TGCGGAACATGGGATACTAGTTTGCGTGCTTGGGTATTC  740 CGTWDTSLNTGVF 1476 TGCGGAACATGGGATACCAGCCTGAATACTGGGGTATTC  741 CGTWDTSLNIWVF 1477 TGCGGAACATGGGATACCAGCCTGAATATTTGGGTGTTC  742 CGTWDTSLNIGVF 1478 TGCGGAACATGGGATACAAGCCTGAATATTGGGGTGTTC  743 CGTWDTSLIAVVF 1479 TGCGGAACATGGGATACCAGCCTGATTGCTGTGGTGTTC  744 CGTWDRSLSGWVF 1480 TGCGGAACGTGGGATAGGAGCCTGAGTGGTTGGGTGTTC  745 CGTWDNRLSGWVF 1481 TGCGGAACATGGGATAACAGGCTGAGTGGTTGGGTGTTC  746 CGTWDKSLSAVVF 1482 TGCGGAACGTGGGATAAGAGCCTGAGTGCTGTGGTCTTC  747 CGTWDKGLSAWVF 1483 TGCGGAACATGGGATAAAGGCCTGAGTGCTTGGGTGTTC  748 CGTWDISLSAGVF 1484 TGCGGAACATGGGATATCAGCCTGAGTGCTGGGGTGTTC  749 CGTWDESLSGGEVVF 1485 TGCGGAACATGGGATGAGAGCCTGAGTGGTGGCGAGGTGGTCTTC  750 CGTWDASLSAWVF 1486 TGCGGAACATGGGATGCCAGCCTGAGTGCCTGGGTGTTC  751 CGTWDAGLSAWVF 1487 TGCGGAACTTGGGATGCCGGCCTGAGTGCTTGGGTGTTC  752 CGAWDTSLSAWVF 1488 TGCGGAGCATGGGATACCAGCCTGAGTGCTTGGGTGTTC  753 CGAWDTSLSAVVF 1489 TGCGGAGCATGGGATACCAGCCTGAGTGCTGTGGTGTTC  754 CGAWDTSLRAGVF 1490 TGCGGAGCATGGGATACCAGCCTGCGTGCTGGGGTTTTC  755 CATWDTSVSAWVF 1491 TGCGCAACATGGGATACCAGCGTGAGTGCTTGGGTGTTC  756 CATWDTSLSAWVF 1492 TGCGCAACATGGGATACCAGCCTGAGTGCGTGGGTGTTC  757 CATWDNTLSAGVF 1493 TGCGCAACATGGGACAACACCCTGAGTGCTGGGGTGTTC  758 CAAWDRSLSVWVF 1494 TGCGCAGCATGGGATAGGAGCCTGAGTGTTTGGGTGTTC  759 CYTWHSSLRGGVF 1495 TGCTACACATGGCATTCCAGTCTGCGTGGTGGGGTGTTC  760 CVTWTSSPSAWVF 1496 TGCGTAACGTGGACTAGTAGCCCGAGTGCTTGGGTGTTC  761 CVTWRGGLVLF 1497 TGCGTGACATGGCGTGGTGGCCTTGTGTTGTTC  762 CVTWDTSLTSVVL 1498 TGCGTAACATGGGATACCAGCCTGACTTCTGTGGTACTC  763 CVTWDTSLSVYWVF 1499 TGCGTAACATGGGATACCAGCCTGAGTGTTTATTGGGTGTTC  764 CVTWDTSLSAWVF 1500 TGCGTTACATGGGATACCAGCCTGAGTGCCTGGGTGTTC  765 CVTWDTDLSVALF 1501 TGCGTCACATGGGATACCGACCTCAGCGTTGCGCTCTTC  766 CVTWDRSLSGWVF 1502 TGCGTAACATGGGATAGGAGCCTGAGTGGTTGGGTGTTC  767 CVTWDRSLREVLF 1503 TGCGTAACATGGGATCGCAGCCTGAGAGAGGTGTTATTC  768 CVTWDRSLRAVVF 1504 TGCGTAACATGGGATCGCAGCCTGAGAGCGGTGGTATTC  769 CVTWDRSLDAGVF 1505 TGCGTAACATGGGACAGGAGCCTCGATGCTGGGGTTTTC  770 CVTWDNTLSAGVF 1506 TGCGTGACATGGGATAACACCCTGAGTGCTGGGGTCTTC  771 CVTWDNNLFGVVF 1507 TGCGTAACATGGGATAACAACCTGTTTGGTGTGGTCTTC  772 CVSWDTSLSGAVF 1508 TGCGTATCATGGGATACCAGCCTGAGTGGTGCGGTATTC  773 CVSWDTSLSAGVF 1509 TGCGTCTCATGGGATACCAGCCTGAGTGCTGGGGTATTC  774 CTTWFRTPSDVVF 1510 TGCACAACATGGTTTAGGACTCCGAGTGATGTGGTCTTC  775 CTTWFRTASDVVF 1511 TGCACAACATGGTTTAGGACTGCGAGTGATGTGGTCTTC  776 CTTWDYGLSVVF 1512 TGCACAACGTGGGATTACGGTCTGAGTGTCGTCTTC  777 CTARDTSLSPGGVF 1513 TGCACAGCAAGGGATACCAGCCTGAGTCCTGGCGGGGTCTTC  778 CSTWNTRPSDVVF 1514 TGCTCAACATGGAATACGAGGCCGAGTGATGTGGTGTTC  779 CSTWESSLTTVVF 1515 TGTTCAACATGGGAGAGCAGTTTGACTACTGTGGTCTTC  780 CSTWDTSLTNVLF 1516 TGCTCAACATGGGATACCAGCCTCACTAATGTGCTATTC  781 CSTWDTSLSGVVF 1517 TGCTCAACATGGGATACCAGCCTGAGTGGAGTAGTCTTC  782 CSTWDHSLKAALF 1518 TGCTCAACATGGGATCACAGCCTGAAAGCTGCACTGTTC  783 CSTWDARLSVRVF 1519 TGCTCAACCTGGGATGCGAGGCTGAGTGTCCGGGTGTTC  784 CSSYTSSSTWVF 1520 TGCTCCTCATATACAAGCAGCAGCACTTGGGTGTTC  785 CSSYATRGLRVLF 1521 TGCAGCTCATACGCAACCCGCGGCCTTCGTGTGTTGTTC  786 CSSWDATLSVRIF 1522 TGTTCATCATGGGACGCCACCCTGAGTGTTCGCATATTC  787 CQVWEGSSDHWVF 1523 TGTCAGGTGTGGGAGGGTAGTAGTGATCATTGGGTGTTC  788 CQTWDNRLSAVVF 1524 TGCCAAACCTGGGATAACAGACTGAGTGCTGTGGTGTTC  789 CQTWDHSLHVGVF 1525 TGTCAAACGTGGGATCACAGCCTGCATGTTGGGGTGTTC  790 CQSYDDILNVWVL 1526 TGCCAGTCCTATGACGACATCTTGAATGTTTGGGTCCTT  791 CNTWDKSLTSELF 1527 TGCAATACATGGGATAAGAGTTTGACTTCTGAACTCTTC  792 CLTWDRSLNVRVF 1528 TGCTTAACATGGGATCGCAGCCTGAATGTGAGGGTGTTC  793 CLTWDHSLTAYVF 1529 TGCCTAACATGGGACCACAGCCTGACTGCTTATGTCTTC  794 CLTRDTSLSAPVF 1530 TGCTTAACAAGGGATACCAGTCTGAGTGCCCCTGTGTTC  795 CKTWESGLNFGHVF 1531 TGCAAAACATGGGAAAGTGGCCTTAATTTTGGCCACGTCTTC  796 CKTWDTSLSAVVF 1532 TGCAAAACATGGGATACCAGCCTGAGTGCTGTGGTCTTC  797 CGVWDVSLGAGVF 1533 TGCGGAGTCTGGGATGTCAGTCTGGGTGCTGGGGTGTTC  798 CGVWDTTPSAVLF 1534 TGCGGAGTCTGGGATACCACCCCGAGTGCCGTTCTTTTC  799 CGVWDTTLSAVLF 1535 TGCGGAGTCTGGGATACCACCCTGAGTGCCGTTCTTTTC  800 CGVWDTSLGVF 1536 TGCGGAGTATGGGATACCAGCCTGGGGGTCTTC  801 CGVWDTNLGKWVF 1537 TGCGGGGTATGGGATACCAACCTGGGTAAATGGGTTTTC  802 CGVWDTGLDAGWVF 1538 TGTGGAGTTTGGGATACTGGCCTGGATGCTGGTTGGGTGTTC  803 CGVWDNVLEAYVF 1539 TGCGGAGTGTGGGATAACGTCCTGGAGGCCTATGTCTTC  804 CGVWDISLSANWVF 1540 TGCGGAGTCTGGGATATCAGCCTGAGTGCTAATTGGGTGTTC  805 CGVWDHSLGIWAF 1541 TGCGGAGTATGGGATCACAGCCTGGGGATTTGGGCCTTC  806 CGVWDDILTAEVF 1542 TGCGGAGTTTGGGATGATATTCTGACTGCTGAAGTGTTC  807 CGVRDTSLGVF 1543 TGCGGAGTTCGGGATACCAGCCTGGGGGTCTTC  808 CGTYDTSLPAWVF 1544 TGCGGAACATACGATACGAGCCTGCCTGCTTGGGTGTTT  809 CGTYDNLVFGYVF 1545 TGCGGAACTTACGATAATCTTGTATTTGGTTATGTCTTC  810 CGTYDDRLREVF 1546 TGCGGAACATACGATGATAGACTCAGAGAGGTGTTC  811 CGTWVTSLSAGVF 1547 TGCGGAACGTGGGTTACCAGCCTGAGTGCTGGGGTGTTC  812 CGTWVSSLTTVVF 1548 TGCGGAACATGGGTTAGCAGCCTGACTACTGTAGTATTC  813 CGTWVSSLNVWVF 1549 TGCGGAACATGGGTTAGCAGCCTGAACGTCTGGGTGTTC  814 CGTWVGRFWVF 1550 TGCGGAACATGGGTTGGCAGGTTTTGGGTATTC  815 CGTWSGGPSGHWLF 1551 TGCGGAACATGGTCTGGCGGCCCGAGTGGCCATTGGTTGTTC  816 CGTWSGGLSGHWLF 1552 TGCGGAACATGGTCTGGCGGCCTGAGTGGCCATTGGTTGTTC  817 CGTWQTGREAVLF 1553 TGCGGAACGTGGCAGACCGGCCGGGAGGCTGTCCTATTT  818 CGTWQSRLRWVF 1554 TGCGGAACGTGGCAGAGCAGGCTGAGGTGGGTGTTC  819 CGTWQSRLGWVF 1555 TGCGGAACGTGGCAGAGCAGGCTGGGGTGGGTGTTC  820 CGTWPRSLSAVWVF 1556 TGCGGAACATGGCCTAGGAGCCTGAGTGCTGTTTGGGTGTTC  821 CGTWNNYLSAGDVVF 1557 TGCGGAACATGGAATAACTACCTGAGTGCTGGCGATGTGGTTTTC  822 CGTWLGSQSPYWVF 1558 TGCGGAACATGGCTTGGCAGCCAGAGTCCTTATTGGGTCTTC  823 CGTWHTGLSAYVF 1559 TGCGGAACATGGCATACCGGCCTGAGTGCTTATGTCTTC  824 CGTWHSTLSAGHWVF 1560 TGCGGAACATGGCATAGTACCCTGAGTGCTGGCCATTGGGTGTTC  825 CGTWHSSLSTWVF 1561 TGCGGAACATGGCATAGTAGCCTGAGTACTTGGGTGTTC  826 CGTWHSSLSAYVF 1562 TGCGGAACATGGCATAGCAGCCTGAGTGCCTATGTCTTC  827 CGTWHSSLSAVVF 1563 TGCGGAACATGGCATAGCAGCCTGAGTGCTGTGGTATTC  828 CGTWHSGLSGWVF 1564 TGCGGAACGTGGCATTCCGGCCTGAGTGGGTGGGTTTTC  829 CGTWHNTLRNVIF 1565 TGCGGAACATGGCATAACACCCTGCGTAATGTGATATTC  830 CGTWHASLTAVF 1566 TGCGGAACATGGCATGCCAGCCTGACTGCTGTGTTC  831 CGTWGWYGSQRGVVF 1567 TGCGGGACATGGGGATGGTATGGCAGCCAGAGAGGCGTCGTCTTC  832 CGTWGWYGGQRGVVF 1568 TGCGGGACATGGGGATGGTATGGCGGCCAGAGAGGCGTCGTCTTC  833 CGTWGTSLSAWVF 1569 TGCGGAACCTGGGGAACCAGCCTGAGTGCTTGGGTGTTC  834 CGTWGSSLTTGLF 1570 TGCGGAACCTGGGGTAGCAGCCTGACTACTGGCCTGTTC  835 CGTWGSSLTAYVF 1571 TGCGGAACATGGGGTAGCAGCCTGACTGCCTATGTCTTC  836 CGTWGSSLSVVF 1572 TGCGGAACATGGGGTAGCAGCCTGAGTGTTGTGTTC  837 CGTWGSSLSGGVF 1573 TGCGGAACATGGGGTAGCAGCCTGAGTGGTGGGGTGTTC  838 CGTWGSSLSAYWVF 1574 TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATTGGGTGTTC  839 CGTWGSSLSAYVVF 1575 TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATGTGGTGTTC  840 CGTWGSSLSAYVF 1576 TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATGTCTTC  841 CGTWGSSLSAVVF 1577 TGCGGAACGTGGGGTAGTAGCCTGAGTGCTGTGGTGTTC  842 CGTWGSSLSAPYVF 1578 TGCGGAACATGGGGTAGCAGCCTGAGTGCTCCTTATGTCTTC  843 CGTWGSSLSAPVF 1579 TGCGGAACATGGGGTAGCAGCCTGAGTGCCCCGGTGTTC  844 CGTWGSSLSAGVF 1580 TGCGGAACATGGGGTAGCAGCCTGAGTGCTGGGGTGTTC  845 CGTWGSSLSAGLF 1581 TGCGGAACTTGGGGTAGCAGCCTGAGTGCTGGACTGTTC  846 CGTWGSSLSAGALF 1582 TGCGGAACATGGGGTAGCAGCCTGAGTGCTGGGGCACTCTTC  847 CGTWGSSLRAWVF 1583 TGCGGAACATGGGGCAGTAGCCTGCGTGCTTGGGTGTTC  848 CGTWFTSLASGVF 1584 TGCGGAACCTGGTTTACTAGTCTGGCTAGTGGGGTTITC  849 CGTWETSLSVVVI 1585 TGCGGAACTTGGGAGACCAGTCTGAGTGTCGTGGTCATC  850 CGTWETSLSGVF 1586 TGCGGAACATGGGAGACCAGCCTGAGTGGTGTCTTC  851 CGTWETSLSDWVF 1587 TGCGGAACATGGGAAACCAGCCTGAGTGATTGGGTATTC  852 CGTWETSLSAGVF 1588 TGCGGAACATGGGAGACCAGCCTGAGTGCTGGGGTATTC  853 CGTWETSLNYVAF 1589 TGCGGAACATGGGAAACCAGCCTTAATTATGTGGCCTTC  854 CGTWETSLNTWLL 1590 TGCGGAACATGGGAGACCAGCCTGAATACTTGGTTGCTC  855 CGTWETSESGNYIF 1591 TGCGGAACATGGGAGACCAGCGAGAGTGGTAATTACATCTTC  856 CGTWETRLGTWVI 1592 TGCGGAACATGGGAAACCAGACTGGGTACTTGGGTGATC  857 CGTWETQLYWVF 1593 TGCGGAACATGGGAGACCCAGTTATATTGGGTGTTC  858 CGTWETGLSAGEVF 1594 TGCGGAACATGGGAGACTGGCCTAAGTGCTGGAGAGGTGTTC  859 CGTWESTLSVFLF 1595 TGCGGAACTTGGGAAAGCACCCTGAGTGTTTTCCTATTC  860 CGTWESSLTVVVF 1596 TGCGGGACATGGGAAAGTAGCCTGACTGTTGTGGTCTTC  861 CGTWESSLTGVVF 1597 TGCGGAACATGGGAAAGTAGCCTGACTGGAGTGGTATTC  862 CGTWESSLTGFVF 1598 TGCGGAACATGGGAAAGCAGCCTGACTGGTTTTGTCTTC  863 CGTWESSLSVGVF 1599 TGTGGAACATGGGAGAGCAGCCTGAGTGTTGGGGTGTTC  864 CGTWESSLSEWVF 1600 TGCGGAACCTGGGAAAGTAGCCTCAGTGAATGGGTGTTC  865 CGTWESSLSAVF 1601 TGCGGAACATGGGAGAGCAGCCTGAGTGCTGTATTC  866 CGTWESSLSAGYIF 1602 TGCGGAACATGGGAGAGCAGCCTGAGTGCTGGTTATATCTTC  867 CGTWESSLSAGVF 1603 TGCGGAACATGGGAGAGCAGCCTGAGTGCTGGAGTGTTC  868 CGTWESSLSAGPVF 1604 TGCGGAACATGGGAAAGCAGCCTGAGCGCTGGCCCGGTGTTC  869 CGTWESSLSAGGQVF 1605 TGCGGAACATGGGAAAGCAGCCTGAGTGCTGGAGGCCAGGTGTTC  870 CGTWESSLSAFGGYVF 1606 TGCGGAACATGGGAGAGCAGCCTGAGTGCCTTCGGCGGTTATGTC TTC  871 CGTWESSLRVWVF 1607 TGCGGAACATGGGAAAGCAGCCTGAGGGTTTGGGTGTTC  872 CGTWESSLFTGPWVF 1608 TGCGGAACATGGGAAAGCAGCCTCTTTACTGGGCCTTGGGTGTTC  873 CGTWESLSATYVF 1609 TGCGGAACATGGGAGAGCCTGAGTGCCACCTATGTCTTC  874 CGTWESGLSAGVF 1610 TGCGGAACATGGGAGAGCGGCCTGAGTGCTGGTGTCTTC  875 CGTWESDFWVF 1611 TGCGGAACATGGGAAAGCGACTTTTGGGTGTTT  876 CGTWENRLSAVVF 1612 TGCGGTACATGGGAAAACAGACTGAGTGCTGTGGTCTTC  877 CGTWENRLSAGVF 1613 TGCGGAACATGGGAAAACAGACTGAGTGCCGGGGTATTC  878 CGTWEISLTTSVVF 1614 TGCGGAACATGGGAAATCAGCCTGACTACTTCTGTGGTATTC  879 CGTWEISLSTSVVF 1615 TGCGGAACATGGGAAATCAGCCTGAGTACTTCTGTGGTATTC  880 CGTWEGSLSVVF 1616 TGCGGAACATGGGAAGGCAGCCTCAGTGTTGTTTTC  881 CGTWEGSLRVF 1617 TGCGGAACATGGGAAGGCAGCCTGAGGGTGTTC  882 CGTWEGSLRHVF 1618 TGCGGAACATGGGAGGGCAGCCTGAGGCACGTGTTC  883 CGTWDYSPVRAGVF 1619 TGCGGAACATGGGATTACAGCCCTGTACGTGCTGGGGTGTTC  884 CGTWDYSLSVYLF 1620 TGCGGAACGTGGGATTACAGCCTGAGTGTTTATCTCTTC  885 CGTWDYSLSSGVVF 1621 TGCGGAACATGGGATTACAGCCTGAGTTCTGGCGTGGTATTC  886 CGTWDYSLSAWVF 1622 TGCGGAACATGGGATTACAGCCTGAGTGCCTGGGTGTTC  887 CGTWDYSLSAEVF 1623 TGCGGAACATGGGATTACAGTCTGAGTGCTGAGGTGTTC  888 CGTWDYSLRRAIF 1624 TGCGGAACATGGGATTACAGCCTGCGTCGTGCGATATTC  889 CGTWDWSLILQLF 1625 TGCGGAACATGGGATTGGAGCCTCATTCTTCAATTGTTC  890 CGTWDVTLHTGVF 1626 TGCGGAACATGGGATGTCACCTTGCATACTGGGGTGTTC  891 CGTWDVTLHIGVF 1627 TGCGGAACATGGGATGTCACCTTGCATATTGGGGTGTTC  892 CGTWDVTLHAGVF 1628 TGCGGAACATGGGATGTCACCTTGCATGCTGGGGTGTTC  893 CGTWDVSLYSGGVF 1629 TGCGGAACATGGGATGTCAGTTTGTATAGTGGCGGGGTCTTC  894 CGTWDVSLTSFVF 1630 TGTGGAACATGGGATGTCAGCCTGACTTCTTTCGTCTTC  895 CGTWDVSLSVGVL 1631 TGCGGAACATGGGATGTCAGCCTGAGTGTTGGGGTGCTC  896 CGTWDVSLSAGDVVF 1632 TGCGGAACGTGGGATGTCAGCCTGAGTGCTGGCGATGTAGTTTTC  897 CGTWDVSLNVVVF 1633 TGCGGAACATGGGATGTCAGCCTGAATGTCGTGGTTTTC  898 CGTWDVSLNTQVF 1634 TGCGGAACATGGGATGTCAGCCTGAATACTCAGGTGTTC  899 CGTWDVSLGALF 1635 TGCGGCACATGGGATGTGAGCCTGGGTGCGCTGTTC  900 CGTWDVNLKTVVF 1636 TGCGGAACGTGGGACGTTAATCTGAAAACTGTCGTTTTC  901 CGTWDVILSAEVF 1637 TGCGGAACATGGGATGTCATCCTGAGTGCTGAGGTATTC  902 CGTWDTTVSAVVF 1638 TGCGGAACATGGGATACCACCGTGAGTGCTGTGGTTTTC  903 CGTWDTTLTAWVF 1639 TGCGGAACATGGGATACCACCCTGACTGCCTGGGTGTTC  904 CGTWDTTLSVFLF 1640 TGCGGAACATGGGACACCACCTTGAGTGTTTTCCTATTC  905 CGTWDTSVSAGVF 1641 TGCGGGACTTGGGATACCAGTGTGAGTGCTGGGGTGTTC  906 CGTWDTSVISWVF 1642 TGCGGAACATGGGATACCAGTGTGATTTCTTGGGTTTTC  907 CGTWDTSRSSLYVVF 1643 TGCGGAACATGGGATACCAGTCGGAGTTCTCTCTATGTGGTCTTC  908 CGTWDTSRSAWVF 1644 TGCGGAACATGGGATACCAGCCGGAGTGCTTGGGTATTC  909 CGTWDTSRNPGGIF 1645 TGCGGAACATGGGATACCAGCCGGAATCCTGGAGGAATTTTC  910 CGTWDTSRGHVF 1646 TGCGGAACATGGGACACCAGTCGGGGTCATGTTTTC  911 CGTWDTSPSTGQVLF 1647 TGCGGAACATGGGATACCAGCCCGAGTACTGGCCAGGTGCTTTTC  912 CGTWDTSPSAWVF 1648 TGCGGAACATGGGATACCAGCCCGAGTGCCTGGGTGTTC  913 CGTWDTSLTWVF 1649 TGCGGAACATGGGATACTAGCCTGACCTGGGTGTTC  914 CGTWDTSLTWFAVF 1650 TGCGGAACATGGGATACCAGCCTGACGTGGTTCGCAGTGTTC  915 CGTWDTSLTVVVF 1651 TGCGGAACATGGGATACCAGCCTGACTGTTGTGGTATTC  916 CGTWDTSLTTSWVF 1652 TGCGGAACATGGGATACCAGCCTGACTACTTCTTGGGTGTTC  917 CGTWDTSLTTGPFWVF 1653 TGCGGAACATGGGATACCAGCCTGACCACTGGTCCTTTTTGGGTGT TC  918 CGTWDTSLTPFYVF 1654 TGCGGAACATGGGATACCAGCCTGACTCCTTTTTATGTCTTC  919 CGTWDTSLTAYVF 1655 TGCGGAACATGGGATACCAGCCTGACTGCTTATGTCTTC  920 CGTWDTSLTAWVF 1656 TGCGGAACATGGGATACCAGCCTGACTGCTTGGGTGTTC  921 CGTWDTSLTAWGVF 1657 TGCGGAACATGGGATACCAGCCTGACTGCGTGGGGGGTGTTC  922 CGTWDTSLTAVVL 1658 TGCGGCACATGGGATACCAGCCTGACTGCGGTGGTTCTC  923 CGTWDTSLTARVF 1659 TGCGGAACCTGGGATACCAGCCTGACTGCTCGGGTTTTC  924 CGTWDTSLTAIVF 1660 TGCGGAACATGGGATACCAGCCTGACTGCGATTGTCTTC  925 CGTWDTSLTAGVF 1661 TGCGGAACATGGGATACCAGCCTGACTGCTGGTGTCTTC  926 CGTWDTSLSVYVF 1662 TGCGGAACATGGGATACCAGCCTGAGTGTTTATGTCTTC  927 CGTWDTSLSVVF 1663 TGCGGAACATGGGATACCAGCCTGAGTGTGGTGTTC  928 CGTWDTSLSVGEF 1664 TGCGGGACATGGGATACCAGCCTGAGTGTTGGGGAATTC  929 CGTWDTSLSTWVF 1665 TGCGGAACATGGGATACCAGCCTGAGTACTTGGGTGTTC  930 CGTWDTSLSTVVF 1666 TGCGGAACATGGGATACCAGCCTGAGTACTGTGGTATTC  931 CGTWDTSLSTGQVLF 1667 TGCGGAACATGGGATACCAGCCTGAGTACTGGCCAGGTGCTTTTC  932 CGTWDTSLSTGPLWVF 1668 TGCGGCACATGGGATACCAGCCTGAGCACTGGTCCTCTTTGGGTGT TC  933 CGTWDTSLSSYVF 1669 TGCGGAACTTGGGATACCAGCCTGAGTTCTTATGTCTTC  934 CGTWDTSLSSVVF 1670 TGCGGAACATGGGATACCAGCCTGAGTTCTGTGGTCTTC  935 CGTWDTSLSSRYIF 1671 TGCGGAACATGGGATACCAGCCTGAGTTCTAGATACATATTC  936 CGTWDTSLSSRFIF 1672 TGCGGAACATGGGATACCAGCCTGAGTTCTAGATTCATATTC  937 CGTWDTSLSSGWVF 1673 TGCGGAACATGGGATACCAGCCTGAGTTCTGGGTGGGTGTTC  938 CGTWDTSLSRYVF 1674 TGCGGAACATGGGATACCAGCCTGAGTCGGTATGTGTTC  939 CGTWDTSLSQWLF 1675 TGCGGAACTTGGGATACCAGTCTGAGTCAATGGCTGTTC  940 CGTWDTSLSPGLWVF 1676 TGCGGAACATGGGATACCAGCCTGAGTCCTGGCCTTTGGGTGTTC  941 CGTWDTSLSNYVF 1677 TGCGGAACATGGGATACCAGCCTGAGTAATTATGTCTTC  942 CGTWDTSLSIWVF 1678 TGCGGAACATGGGATACCAGCCTAAGTATTTGGGTGTTC  943 CGTWDTSLSIGPFWVF 1679 TGCGGCACATGGGATACCAGCCTGAGCATTGGTCCTTTTTGGGTGT TC  944 CGTWDTSLSGWVF 1680 TGCGGAACATGGGATACCAGCCTGAGTGGTTGGGTGTTC  945 CGTWDTSLSGTVF 1681 TGCGGAACATGGGATACCAGCCTGAGTGGTACAGTGTTC  946 CGTWDTSLSGGQVF 1682 TGCGGAACATGGGATACTAGTCTGAGTGGTGGCCAGGTGTTC  947 CGTWDTSLSGGIF 1683 TGCGGAACATGGGATACCAGCCTGAGTGGTGGGATATTC  948 CGTWDTSLSGEDVVI 1684 TGCGGAACATGGGATACCAGCCTGAGTGGTGAGGATGTGGTAATC  949 CGTWDTSLSFLYAF 1685 TGCGGAACATGGGATACCAGCCTGAGTTTCCTTTATGCTTTC  950 CGTWDTSLSEVVF 1686 TGCGGAACATGGGATACCAGCCTGAGTGAGGTCGTATTC  951 CGTWDTSLSEVF 1687 TGCGGAACATGGGATACCAGCCTGAGTGAAGTGTTC  952 CGTWDTSLSENWVF 1688 TGCGGAACATGGGATACTAGCCTGAGTGAAAATTGGGTGTTC  953 CGTWDTSLSAYIF 1689 TGCGGAACATGGGATACCAGCCTGAGTGCCTACATATTC  954 CGTWDTSLSAVVL 1690 TGCGGAACATGGGATACCAGCCTGAGTGCTGTGGTACTC  955 CGTWDTSLSAVF 1691 TGCGGAACATGGGATACCAGCCTGAGTGCTGTTTTC  956 CGTWDTSLSARVF 1692 TGCGGAACATGGGATACCAGCCTGAGTGCCCGGGTGTTC  957 CGTWDTSLSARQVF 1693 TGCGGCACATGGGATACCAGCCTGAGTGCCCGCCAGGTATTC  958 CGTWDTSLSALVF 1694 TGCGGAACATGGGATACCAGCCTGAGTGCTTTGGTTTTC  959 CGTWDTSLSAKVF 1695 TGCGGAACATGGGATACCAGCCTGAGTGCTAAGGTGTTC  960 CGTWDTSLSAKIF 1696 TGCGGAACATGGGATACCAGCCTGAGTGCGAAAATCTTC  961 CGTWDTSLSAKAVF 1697 TGCGGAACATGGGATACCAGCCTGAGTGCCAAGGCGGTATTC  962 CGTWDTSLSAHAVF 1698 TGCGGAACATGGGATACCAGCCTGAGTGCCCATGCTGTGTTC  963 CGTWDTSLSAGYVF 1699 TGCGGAACATGGGATACCAGCCTGAGTGCTGGCTATGTCTTC  964 CGTWDTSLSAGRWVF 1700 TGCGGAACATGGGACACCAGTCTGAGTGCTGGCCGCTGGGTGTTC  965 CGTWDTSLSAGIF 1701 TGCGGAACATGGGATACCAGCCTGAGTGCTGGGATATTC  966 CGTWDTSLSAGGFRVF 1702 TGCGGAACATGGGATACCAGCCTGAGTGCTGGTGGGTTCCGGGTC TTC  967 CGTWDTSLSAGAF 1703 TGCGGAACATGGGATACCAGCCTGAGTGCTGGGGCATTC  968 CGTWDTSLSADWFF 1704 TGCGGAACATGGGATACCAGTCTGAGTGCTGATTGGTTTTTC  969 CGTWDTSLSADEYVF 1705 TGCGGAACATGGGATACCAGCCTGAGTGCTGATGAATATGTCTTC  970 CGTWDTSLSAAWVF 1706 TGCGGCACATGGGATACCAGCCTGAGTGCGGCTTGGGTGTTC  971 CGTWDTSLSAALF 1707 TGCGGAACATGGGATACCAGCCTGAGTGCTGCGCTATTC  972 CGTWDTSLSAAGVF 1708 TGCGGAACATGGGATACCAGCCTGAGTGCTGCGGGGGTTTTC  973 CGTWDTSLRVVVF 1709 TGCGGAACATGGGATACCAGCCTGAGAGTTGTGGTTTTC  974 CGTWDTSLRTWVF 1710 TGCGGAACATGGGATACCAGCCTGAGAACCTGGGTATTC  975 CGTWDTSLRGAVF 1711 TGCGGAACGTGGGATACCAGCCTGAGGGGTGCAGTGTTC  976 CGTWDTSLRAVVF 1712 TGCGGAACATGGGATACCAGCCTGCGTGCTGTGGTATTC  977 CGTWDTSLNVVYVF 1713 TGCGGAACATGGGATACAAGCCTGAATGTAGTTTATGTCTTC  978 CGTWDTSLNTYLF 1714 TGCGGAACATGGGATACCAGCCTCAACACCTACCTGTTC  979 CGTWDTSLNFAWLF 1715 TGCGGAACATGGGATACTAGCCTGAACTTCGCTTGGCTGTTC  980 CGTWDTSLLVWLF 1716 TGCGGCACATGGGATACCAGCCTTCTTGTGTGGCTTTTC  981 CGTWDTSLKTWVF 1717 TGCGGAACATGGGATACCAGTCTGAAGACGTGGGTGTTC  982 CGTWDTSLIVWVF 1718 TGCGGAACATGGGATACCAGTCTGATTGTCTGGGTGTTC  983 CGTWDTSLITGVF 1719 TGCGGAACATGGGATACCAGCCTAATTACTGGGGTGTTC  984 CGTWDTSLISVVF 1720 TGCGGAACATGGGATACCAGCCTGATTAGCGTGGTATTC  985 CGTWDTSLIAYVF 1721 TGCGGAACATGGGATACCAGCCTGATTGCTTATGTCTTC  986 CGTWDTSLHTELF 1722 TGCGGAACATGGGATACCAGCCTGCACACTGAGTTGTTC  987 CGTWDTSLGSYVF 1723 TGCGGAACTTGGGATACCAGCCTGGGTTCTTATGTCTTC  988 CGTWDTSLGSLWVF 1724 TGCGGAACATGGGATACCAGCCTGGGTTCTCTTTGGGTGTTC  989 CGTWDTSLGSGVF 1725 TGCGGTACATGGGATACCAGCCTGGGTTCTGGGGTATTC  990 CGTWDTSLGGRGVF 1726 TGCGGAACTTGGGATACCAGTCTGGGTGGTAGAGGGGTCTTC  991 CGTWDTSLGAWVF 1727 TGCGGAACATGGGATACCAGCCTGGGTGCTTGGGTGTTC  992 CGTWDTSLGAVVF 1728 TGCGGAACATGGGATACCAGCCTGGGTGCCGTGGTATTC  993 CGTWDTSLGAGVF 1729 TGCGGAACATGGGATACCAGCCTGGGTGCTGGGGTATTC  994 CGTWDTSLGAGLF 1730 TGCGGAACATGGGATACCAGCCTGGGTGCTGGCCTATTC  995 CGTWDTSLDAVVF 1731 TGCGGAACATGGGATACCAGTCTGGATGCTGTGGTTTTC  996 CGTWDTSLDAVLF 1732 TGCGGGACTTGGGATACCAGCCTGGATGCTGTGCTGTTC  997 CGTWDTSLAWVF 1733 TGCGGAACATGGGATACCAGCCTGGCTTGGGTGTTC  998 CGTWDTSLATGLF 1734 TGCGGAACATGGGATACCAGCCTGGCGACTGGACTGTTC  999 CGTWDTSLAPVVF 1735 TGCGGGACATGGGATACCAGCCTGGCCCCTGTAGTCTTC 1000 CGTWDTRLTIVIF 1736 TGCGGAACATGGGACACCCGCCTGACTATTGTGATCTTC 1001 CGTWDTRLSVWLF 1737 TGTGGAACATGGGACACCAGGCTGAGTGTTTGGCTGTTC 1002 CGTWDTRLSVGVF 1738 TGCGGAACGTGGGACACCAGACTGAGTGTTGGGGTTTTC 1003 CGTWDTRLSTVIF 1739 TGCGGCACATGGGATACCAGACTGAGTACTGTAATTTTC 1004 CGTWDTRLSSVVF 1740 TGCGGAACATGGGATACCCGCCTGAGTTCTGTGGTCTTC 1005 CGTWDTRLSIVVF 1741 TGCGGAACATGGGATACCCGCCTGAGTATTGTGGTTTTC 1006 CGTWDTRLSAYVVF 1742 TGCGGAACATGGGATACCAGACTGAGTGCCTATGTGGTATTC 1007 CGTWDTRLSAWVF 1743 TGCGGAACCTGGGACACCCGCCTGAGTGCGTGGGTGTTC 1008 CGTWDTRLSAVVF 1744 TGCGGAACATGGGATACCAGACTGAGTGCTGTGGTGTTC 1009 CGTWDTRLSAGLF 1745 TGCGGAACATGGGATACCCGCCTGAGTGCTGGGTTGTTC 1010 CGTWDTRLSAGGVF 1746 TGCGGAACATGGGATACCAGACTGAGTGCTGGTGGGGTGTTC 1011 CGTWDTRLNVWLF 1747 TGCGGAACATGGGATACCAGATTGAATGTGTGGCTATTC 1012 CGTWDTNREVVLL 1748 TGCGGAACATGGGATACCAACCGGGAAGTTGTGCTCCTC 1013 CGTWDTNLRAHVF 1749 TGCGGAACATGGGATACCAACCTGCGTGCCCATGTCTTC 1014 CGTWDTNLPAVVF 1750 TGCGGAACATGGGATACTAATCTGCCCGCTGTAGTGTTC 1015 CGTWDTNLGGVF 1751 TGCGGAACATGGGACACCAATTTGGGTGGGGTGTTC 1016 CGTWDTIVSIGVF 1752 TGCGGAACATGGGATACCATCGTGAGTATTGGGGTGTTC 1017 CGTWDTILSAVVF 1753 TGCGGAACATGGGATACCATCCTGAGTGCGGTGGTGTTC 1018 CGTWDTILSAEVF 1754 TGCGGCACATGGGATACCATCCTGAGTGCTGAGGTGTTC 1019 CGTWDTHLGVVF 1755 TGCGGAACATGGGATACCCACCTGGGTGTGGTTTTC 1020 CGTWDTGPSPHWLF 1756 TGCGGAACATGGGATACCGGCCCGAGCCCTCATTGGCTGTTC 1021 CGTWDTGLTFGGVF 1757 TGCGGAACATGGGATACCGGCCTGACTTTTGGAGGCGTGTTC 1022 CGTWDTGLTAFVF 1758 TGCGGAACATGGGATACCGGCCTGACTGCTTTTGTCTTC 1023 CGTWDTGLSVWVF 1759 TGCGGAACATGGGATACCGGCCTGAGTGTTTGGGTGTTC 1024 CGTWDTGLSTGIF 1760 TGCGGAACATGGGATACCGGCCTGAGTACTGGGATTTTC 1025 CGTWDTGLSSLLF 1761 TGCGGAACATGGGATACCGGCCTGAGTTCCCTGCTCTTC 1026 CGTWDTGLSIVVF 1762 TGCGGAACGTGGGACACCGGCCTGAGTATTGTGGTGTTC 1027 CGTWDTGLSFVVF 1763 TGCGGAACGTGGGACACCGGCCTGAGTTTTGTGGTGTTC 1028 CGTWDTGLSAWVF 1764 TGCGGAACATGGGATACCGGCCTGAGTGCTTGGGTGTTC 1029 CGTWDTGLSAGVVF 1765 TGCGGAACATGGGATACCGGCCTGAGTGCTGGTGTGGTATTC 1030 CGTWDTGLRGWIF 1766 TGCGGAACATGGGATACCGGTCTGAGGGGTTGGATTTTC 1031 CGTWDTELSAGVF 1767 TGCGGAACATGGGATACCGAGCTAAGTGCGGGGGTCTTC 1032 CGTWDTALTAGVF 1768 TGCGGAACGTGGGATACCGCCCTGACTGCTGGGGTGTTC 1033 CGTWDTALSLVVF 1769 TGCGGAACATGGGATACTGCCCTGAGTCTTGTGGTCTTC 1034 CGTWDTALSAWLF 1770 TGCGGAACATGGGATACCGCCCTGAGTGCCTGGCTGTTC 1035 CGTWDTALSAGVF 1771 TGCGGCACATGGGATACCGCCCTGAGTGCTGGGGTGTTC 1036 CGTWDTALRGVLF 1772 TGCGGAACATGGGATACCGCCCTGCGTGGCGTGCTGTTC 1037 CGTWDTALKEWLF 1773 TGCGGAACATGGGATACCGCCCTGAAAGAATGGCTGTTC 1038 CGTWDRTLTAGDVLF 1774 TGCGGAACATGGGATAGGACCCTGACTGCTGGCGATGTGCTCTTC 1039 CGTWDRSVTYVF 1775 TGCGGAACATGGGATAGAAGCGTGACTTATGTCTTC 1040 CGTWDRSRNEWVF 1776 TGCGGAACATGGGATCGCAGCCGAAATGAATGGGTGTTC 1041 CGTWDRSLTVWVF 1777 TGCGGAACATGGGATCGCAGTCTGACTGTTTGGGTCTTC 1042 CGTWDRSLTPGWLF 1778 TGCGGAACATGGGATCGCAGCCTGACTCCTGGGTGGTTGTTC 1043 CGTWDRSLTAWVF 1779 TGCGGAACATGGGATAGAAGCCTGACTGCTTGGGTGTTC 1044 CGTWDRSLSVVVF 1780 TGCGGAACATGGGACCGCAGCCTGAGTGTTGTGGTATTC 1045 CGTWDRSLSVVF 1781 TGCGGCACATGGGATCGCAGCCTGAGTGTAGTCTTC 1046 CGTWDRSLSVQLF 1782 TGCGGAACATGGGATAGGAGCCTGAGTGTTCAATTGTTC 1047 CGTWDRSLSVLWVF 1783 TGCGGAACATGGGATCGCAGCCTCAGTGTTCTTTGGGTGTTC 1048 CGTWDRSLSVGLF 1784 TGCGGAACATGGGATCGCAGCCTGAGTGTTGGATTATTC 1049 CGTWDRSLSTWVF 1785 TGCGGAACATGGGATCGCAGCCTGAGTACTTGGGTGTTC 1050 CGTWDRSLSTHWVL 1786 TGCGGAACATGGGATAGAAGCCTGAGTACTCATTGGGTGCTC 1051 CGTWDRSLSTHWVF 1787 TGCGGAACATGGGATAGAAGCCTGAGTACTCATTGGGTGTTC 1052 CGTWDRSLSSAVF 1788 TGCGGAACCTGGGATCGAAGCCTGAGTTCTGCGGTGTTC 1053 CGTWDRSLSPSYVF 1789 TGCGGAACATGGGACAGAAGCCTGAGTCCCTCTTATGTCTTC 1054 CGTWDRSLSGEVF 1790 TGCGGAACATGGGATAGGAGCCTGAGTGGTGAGGTGTTC 1055 CGTWDRSLSGAVF 1791 TGCGGAACATGGGATAGGAGCCTGAGTGGTGCGGTGTTC 1056 CGTWDRSLSAVAF 1792 TGCGGAACATGGGATCGCAGCCTGAGTGCTGTGGCATTC 1057 CGTWDRSLSAGGEF 1793 TGCGGAACATGGGATAGGAGCCTGAGTGCCGGGGGGGAATTC 1058 CGTWDRSLSAFWVF 1794 TGCGGAACATGGGATCGCAGCCTGAGTGCTTTTTGGGTGTTC 1059 CGTWDRSLSAAVF 1795 TGCGGAACATGGGATAGGAGCCTGAGTGCTGCGGTGTTC 1060 CGTWDRSLSAALF 1796 TGCGGAACATGGGATAGGAGCCTGAGTGCTGCACTCTTC 1061 CGTWDRSLRVF 1797 TGCGGAACATGGGATCGCAGCCTGAGAGTGTTC 1062 CGTWDRSLNWVF 1798 TGCGGTACATGGGACAGAAGCCTTAATTGGGTGTTC 1063 CGTWDRSLNVYVF 1799 TGCGGAACATGGGATCGCAGCCTGAATGTTTATGTCTTC 1064 CGTWDRSLNVGVF 1800 TGCGGAACATGGGATAGGAGCCTGAATGTTGGGGTGTTC 1065 CGTWDRSLHVVF 1801 TGCGGAACATGGGATCGGAGCCTGCATGTGGTCTTC 1066 CGTWDRSLGGWVF 1802 TGTGGAACATGGGATCGCAGCCTGGGTGGTTGGGTGTTC 1067 CGTWDRSLGAFWVF 1803 TGCGGAACATGGGATCGCAGCCTGGGTGCTTTTTGGGTGTTC 1068 CGTWDRSLFWVF 1804 TGCGGAACATGGGATAGAAGCCTGTTTTGGGTGTTC 1069 CGTWDRSLAAGVF 1805 TGCGGAACGTGGGATCGCAGCCTGGCTGCTGGGGTGTTC 1070 CGTWDRRLSGVVF 1806 TGCGGAACATGGGATAGGAGGTTGAGTGGTGTCGTATTC 1071 CGTWDRRLSDVVF 1807 TGCGGAACGTGGGATCGCCGCCTAAGTGATGTGGTATTC 1072 CGTWDRRLSAVVF 1808 TGCGGAACATGGGATAGGAGGCTGAGTGCTGTGGTATTC 1073 CGTWDRRLNVAFF 1809 TGCGGAACATGGGATAGACGCCTGAATGTTGCGTTCTTC 1074 CGTWDRRLLAVF 1810 TGTGGAACATGGGATAGGAGGCTGCTTGCTGTTTTC 1075 CGTWDRNLRAVVF 1811 TGCGGAACTTGGGATAGGAACCTGCGCGCCGTGGTCTTC 1076 CGTWDRLSAGVF 1812 TGCGGAACATGGGATAGGCTGAGTGCTGGGGTGTTC 1077 CGTWDRGPNTGVF 1813 TGCGGAACATGGGATAGAGGCCCGAATACTGGGGTATTC 1078 CGTWDRGLNTVYVF 1814 TGCGGAACATGGGATAGAGGCCTGAATACTGTTTACGTCTTC 1079 CGTWDNYVSAPWVF 1815 TGCGGAACATGGGATAACTATGTGAGTGCCCCTTGGGTGTTC 1080 CGTWDNYLSAGDVVF 1816 TGCGGAACATGGGATAACTACCTGAGTGCTGGCGATGTGGTTTTC 1081 CGTWDNYLRAGVF 1817 TGCGGAACATGGGATAACTACCTGAGAGCTGGGGTCTTC 1082 CGTWDNYLGAVVF 1818 TGCGGAACATGGGACAATTATCTGGGTGCCGTGGTTTTC 1083 CGTWDNYLGAGVF 1819 TGCGGAACATGGGATAACTACCTGGGTGCGGGGGTGTTC 1084 CGTWDNTVSAPWVF 1820 TGCGGAACATGGGATAACACCGTGAGTGCCCCTTGGGTTTTC 1085 CGTWDNTLSLWVF 1821 TGCGGAACATGGGATAACACCCTGAGTCTTTGGGTGTTC 1086 CGTWDNTLSAGVF 1822 TGCGGAACATGGGATAACACCCTGAGTGCTGGGGTCTTC 1087 CGTWDNTLLTVLF 1823 TGCGGAACATGGGACAACACTCTGCTTACTGTGTTATTC 1088 CGTWDNRLSSVIF 1824 TGCGGAACATGGGATAACAGACTGAGTAGTGTGATTTTC 1089 CGTWDNRLSAVVF 1825 TGCGGAACATGGGATAACAGGTTGAGTGCTGTGGTCTTC 1090 CGTWDNRLSAGGIF 1826 TGCGGAACATGGGATAACAGGCTGAGTGCTGGTGGGATATTC 1091 CGTWDNRLSAEVF 1827 TGCGGAACATGGGATAACAGACTGAGTGCTGAGGTGTTC 1092 CGTWDNRLRVGVL 1828 TGTGGAACATGGGATAACAGACTGCGTGTTGGGGTTCTC 1093 CGTWDNRLLENVF 1829 TGCGGAACATGGGATAATCGCCTGCTTGAGAATGTCTTC 1094 CGTWDNNLRAVF 1830 TGCGGAACATGGGATAACAACCTGCGTGCTGTCTTC 1095 CGTWDNNLRAGVF 1831 TGCGGAACTTGGGATAATAACCTGCGTGCTGGAGTGTTC 1096 CGTWDNNLGGGRVF 1832 TGCGGAACATGGGACAACAATTTGGGCGGTGGCCGGGTGTTC 1097 CGTWDNNLGAGVL 1833 TGCGGAACATGGGATAACAACCTGGGTGCTGGCGTCCTC 1098 CGTWDNNLGAGVF 1834 TGCGGAACATGGGATAACAACCTGGGTGCTGGCGTCTTC 1099 CGTWDNILSAAVF 1835 TGCGGAACTTGGGATAACATCCTGAGCGCTGCGGTGTTC 1100 CGTWDNILDAGVF 1836 TGCGGAACCTGGGATAACATCTTGGATGCAGGGGTTTTC 1101 CGTWDNDLSGWLF 1837 TGCGGAACATGGGATAACGACCTGAGTGGTTGGCTGTTC 1102 CGTWDNDLSAWVF 1838 TGCGGAACATGGGATAACGACCTGAGTGCCTGGGTGTTC 1103 CGTWDLTLGGVVF 1839 TGCGGAACATGGGATCTCACCCTGGGTGGTGTGGTGTTC 1104 CGTWDLSLSAGVF 1840 TGCGGAACATGGGATCTCAGCCTGAGTGCTGGGGTATTC 1105 CGTWDLSLKEWVF 1841 TGCGGAACATGGGATCTCAGCCTGAAAGAATGGGTGTTC 1106 CGTWDLSLDAVVF 1842 TGCGGAACGTGGGATCTCAGCCTGGATGCTGTTGTTTTC 1107 CGTWDLKVF 1843 TGCGGAACCTGGGACCTGAAGGTTTTC 1108 CGTWDKTLSVWVF 1844 TGCGGAACATGGGATAAGACTCTGAGTGTTTGGGTGTTC 1109 CGTWDKSLSVWVF 1845 TGCGGAACATGGGATAAGAGCCTGAGTGTTTGGGTGTTC 1110 CGTWDKSLSGVVF 1846 TGCGGAACATGGGATAAGAGCCTGAGTGGTGTGGTATTT 1111 CGTWDKSLSDWVF 1847 TGCGGAACATGGGATAAGAGCCTGAGTGATTGGGTGTTC 1112 CGTWDKSLSALVF 1848 TGCGGAACATGGGATAAGAGCCTGAGTGCTTTGGTTTTC 1113 CGTWDKSLSAGVF 1849 TGCGGAACATGGGATAAGAGCCTGAGTGCTGGCGTCTTC 1114 CGTWDKSLSADVF 1850 TGCGGAACATGGGATAAGAGCCTGAGTGCCGACGTCTTC 1115 CGTWDKRLTIVVF 1851 TGCGGAACATGGGATAAACGCCTGACTATTGTGGTCTTC 1116 CGTWDKRLSAWVL 1852 TGCGGAACATGGGATAAACGCCTGAGTGCCTGGGTGCTC 1117 CGTWDKNLRAVVF 1853 TGCGGAACATGGGATAAGAACCTGCGTGCTGTGGTCTTC 1118 CGTWDITLSGFVF 1854 TGCGGAACATGGGATATCACCCTGAGTGGGTTTGTCTTC 1119 CGTWDITLHTGVF 1855 TGCGGAACATGGGATATCACCTTGCATACTGGAGTATTC 1120 CGTWDISVTVVF 1856 TGCGGAACATGGGATATCAGTGTGACTGTGGTGTTC 1121 CGTWDISVRGYAF 1857 TGCGGAACATGGGATATCAGTGTGAGGGGTTATGCCTTC 1122 CGTWDISRWVF 1858 TGCGGAACATGGGATATCAGCCGTTGGGTTTTC 1123 CGTWDISPSAWVF 1859 TGCGGAACATGGGATATCAGCCCGAGTGCTTGGGTGTTC 1124 CGTWDISLSVWVF 1860 TGCGGAACATGGGATATTAGCCTGAGTGTCTGGGTGTTC 1125 CGTWDISLSVVF 1861 TGCGGAACATGGGATATCAGCCTGAGTGTTGTATTC 1126 CGTWDISLSSVVF 1862 TGCGGAACTTGGGATATCAGCCTGAGTTCTGTGGTGTTC 1127 CGTWDISLSHWLF 1863 TGCGGAACATGGGATATCAGCCTGAGTCACTGGTTGTTC 1128 CGTWDISLSGWVF 1864 TGCGGAACATGGGATATCAGTCTGAGTGGTTGGGTGTTC 1129 CGTWDISLSGRVF 1865 TGCGGAACATGGGATATCAGCCTGAGTGGTCGAGTGTTC 1130 CGTWDISLSAWAF 1866 TGCGGAACATGGGACATCAGCCTGAGTGCTTGGGCGTTC 1131 CGTWDISLSAVVF 1867 TGCGGAACATGGGATATCAGCCTGAGTGCTGTGGTTTTC 1132 CGTWDISLSAVIF 1868 TGCGGGACATGGGACATCAGCCTGAGTGCTGTGATATTC 1133 CGTWDISLSAVF 1869 TGCGGAACATGGGATATCAGCCTGAGTGCTGTGTTC 1134 CGTWDISLSARVF 1870 TGCGGAACATGGGATATCAGCCTGAGTGCCCGGGTGTTC 1135 CGTWDISLSALVF 1871 TGCGGAACATGGGATATCAGCCTGAGTGCCCTGGTGTTC 1136 CGTWDISLSAHVF 1872 TGCGGAACATGGGATATTAGCCTGAGTGCCCATGTCTTC 1137 CGTWDISLSAGVVF 1873 TGCGGAACATGGGATATCAGCCTGAGTGCTGGGGTGGTATTC 1138 CGTWDISLSAGPYVF 1874 TGCGGAACATGGGATATCAGCCTGAGTGCCGGCCCTTATGTCTTC 1139 CGTWDISLSAGGVF 1875 TGCGGCACATGGGATATCAGCCTGAGTGCTGGAGGGGTGTTC 1140 CGTWDISLSAEVF 1876 TGCGGAACATGGGATATCAGCCTGAGTGCTGAGGTTTTC 1141 CGTWDISLSAAVF 1877 TGCGGAACATGGGATATCAGCCTGAGTGCTGCTGTGTTC 1142 CGTWDISLRAVF 1878 TGCGGAACATGGGATATCAGCCTGCGTGCTGTGTTC 1143 CGTWDISLNTGVF 1879 TGCGGAACATGGGATATTAGCCTGAATACTGGGGTGTTC 1144 CGTWDISLNNYVF 1880 TGCGGAACATGGGATATCAGCCTAAATAATTATGTCTTC 1145 CGTWDISLIAGVF 1881 TGCGGAACATGGGATATCAGCCTAATTGCTGGGGTATTC 1146 CGTWDISLHTWLF 1882 TGCGGAACATGGGATATCAGCCTGCATACTTGGCTGTTC 1147 CGTWDIRLTDELLF 1883 TGCGGAACATGGGATATCCGCCTGACCGATGAGCTGTTATTC 1148 CGTWDIRLSGFVF 1884 TGCGGAACATGGGATATCAGACTGAGCGGTTTTGTTTTC 1149 CGTWDINLGAGGLYVF 1885 TGCGGAACATGGGATATCAACCTGGGTGCTGGGGGCCTTTATGTC TTC 1150 CGTWDIILSAEVF 1886 TGCGGAACATGGGATATCATCCTGAGTGCTGAGGTATTC 1151 CGTWDHTLSAVF 1887 TGCGGAACATGGGATCACACCCTGAGTGCTGTCTTC 1152 CGTWDHTLLTVLF 1888 TGCGGAACATGGGACCACACTCTGCTTACTGTGTTATTC 1153 CGTWDHSLTAVVF 1889 TGCGGAACATGGGATCACAGCCTGACTGCTGTGGTATTC 1154 CGTWDHSLTAGIF 1890 TGCGGAACCTGGGATCACAGCCTGACTGCTGGGATATTC 1155 CGTWDHSLSVVLF 1891 TGCGGAACATGGGATCACAGCCTGAGTGTTGTATTATTC 1156 CGTWDHSLSLVF 1892 TGCGGAACATGGGATCACAGCCTGAGTTTGGTATTC 1157 CGTWDHSLSIGVF 1893 TGCGGAACATGGGATCACAGCCTGTCTATTGGGGTTTTC 1158 CGTWDHSLSAGVF 1894 TGCGGAACATGGGATCACAGCCTGAGTGCTGGGGTGTTC 1159 CGTWDHSLSAFVF 1895 TGTGGAACTTGGGATCACAGCCTGAGTGCTTTCGTGTTC 1160 CGTWDHSLSAAVF 1896 TGCGGAACATGGGATCACAGTCTGAGTGCTGCTGTTTTC 1161 CGTWDHNLRAVF 1897 TGCGGAACATGGGACCACAATCTGCGTGCTGTCTTC 1162 CGTWDFTLSVGRF 1898 TGCGGGACATGGGATTTCACCCTGAGTGTTGGGCGCTTC 1163 CGTWDFTLSAPVF 1899 TGCGGAACATGGGATTTCACCCTGAGTGCTCCTGTCTTC 1164 CGTWDFSVSAGWVF 1900 TGCGGAACGTGGGATTTCAGCGTGAGTGCTGGGTGGGTGTTC 1165 CGTWDFSLTTWLF 1901 TGCGGAACGTGGGATTTCAGTCTTACTACCTGGTTATTC 1166 CGTWDFSLSVWVF 1902 TGCGGAACATGGGATTTCAGCCTGAGTGTTTGGGTGTTC 1167 CGTWDFSLSTGVF 1903 TGCGGAACATGGGATTTCAGCCTGAGTACTGGGGTTTTC 1168 CGTWDFSLSGVVF 1904 TGCGGCACATGGGATTTCAGCCTGAGTGGTGTGGTATTC 1169 CGTWDFSLSGFVF 1905 TGCGGAACATGGGATTTCAGCCTGAGTGGTTTCGTGTTC 1170 CGTWDFSLSAGVF 1906 TGCGGAACATGGGATTTCAGCCTGAGTGCTGGGGTGTTC 1171 CGTWDETVRGWVF 1907 TGCGGAACATGGGATGAAACCGTGAGAGGTTGGGTGTTC 1172 CGTWDESLRSWVF 1908 TGCGGAACATGGGATGAAAGTCTGAGAAGCTGGGTGTTC 1173 CGTWDERQTDESYVF 1909 TGCGGAACTTGGGATGAGAGGCAGACTGATGAGTCCTATGTCTTC 1174 CGTWDERLVAGQVF 1910 TGCGGAACATGGGATGAGAGACTCGTTGCTGGCCAGGTCTTC 1175 CGTWDERLSPGAFF 1911 TGCGGAACATGGGATGAGAGACTGAGTCCTGGAGCTTTTTTC 1176 CGTWDEKVF 1912 TGCGGAACATGGGATGAGAAGGTGTTC 1177 CGTWDEGQTTDFFVF 1913 TGCGGAACCTGGGATGAAGGCCAGACTACTGATTTCTTTGTCTTC 1178 CGTWDDTLAGVVF 1914 TGCGGAACATGGGATGACACCCTGGCTGGTGTGGTCTTC 1179 CGTWDDRLTSAVF 1915 TGCGGAACATGGGATGACAGGCTGACTTCTGCGGTCTTC 1180 CGTWDDRLFVVVF 1916 TGCGGAACATGGGATGACAGACTGTTTGTTGTGGTATTC 1181 CGTWDDNLRGWVF 1917 TGCGGAACATGGGATGATAACCTGAGAGGTTGGGTGTTC 1182 CGTWDDNLRGVVF 1918 TGCGGAACATGGGATGACAACCTGCGTGGTGTCGTGTTC 1183 CGTWDDNLNIGRVF 1919 TGCGGAACCTGGGATGACAATTTGAATATTGGAAGGGTGTTC 1184 CGTWDDILSAVIF 1920 TGCGGAACATGGGATGACATCCTGAGTGCTGTGATATTC 1185 CGTWDDILRGWVF 1921 TGCGGAACATGGGATGATATCCTGAGAGGTTGGGTGTTC 1186 CGTWDATLSPGWLF 1922 TGCGGAACATGGGATGCCACCCTGAGTCCTGGGTGGTTATTC 1187 CGTWDASVTSWVF 1923 TGCGGAACATGGGATGCCAGCGTGACTTCTTGGGTGTTC 1188 CGTWDASLTSVVF 1924 TGCGGAACATGGGATGCCAGCCTGACTTCTGTGGTCTTC 1189 CGTWDASLSVWVF 1925 TGCGGAACATGGGATGCCAGCCTGAGTGTTTGGGTGTTC 1190 CGTWDASLSVPWVF 1926 TGCGGAACATGGGATGCCAGCCTGAGTGTTCCTTGGGTGTTC 1191 CGTWDASLSVAVF 1927 TGCGGAACATGGGATGCCAGCCTGAGTGTGGCGGTATTC 1192 CGTWDASLSTWVF 1928 TGCGGAACATGGGATGCCAGCCTGAGTACCTGGGTATTC 1193 CGTWDASLSGVVF 1929 TGCGGAACATGGGATGCCAGCCTGAGTGGTGTGGTATTC 1194 CGTWDASLSGGGEF 1930 TGCGGAACATGGGATGCCAGCCTGAGTGGTGGGGGAGAATTC 1195 CGTWDASLSAGVF 1931 TGCGGAACATGGGATGCCAGCCTGAGTGCTGGGGTGTTC 1196 CGTWDASLSAGLF 1932 TGCGGAACATGGGATGCCAGCCTGAGTGCTGGGCTTTTC 1197 CGTWDASLSAEVF 1933 TGTGGCACATGGGATGCCAGCCTGAGTGCTGAAGTCTTC 1198 CGTWDASLSADFWVF 1934 TGCGGAACATGGGATGCCAGCCTGAGTGCTGACTTTTGGGTGTTC 1199 CGTWDASLRVFF 1935 TGCGGAACATGGGATGCCAGCCTGAGAGTCTTCTTC 1200 CGTWDASLRAVVL 1936 TGCGGAACATGGGATGCCAGTCTGAGGGCTGTGGTACTC 1201 CGTWDASLNIWVF 1937 TGCGGAACATGGGATGCCAGCCTGAATATTTGGGTTTTC 1202 CGTWDASLKNLVF 1938 TGCGGGACATGGGATGCCAGCCTGAAGAATCTGGTCTTC 2322 CGTWDASLGAWVF 1939 TGCGGAACATGGGATGCCAGCCTGGGTGCCTGGGTATTC 2323 CGTWDASLGAVVF 1940 TGCGGAACATGGGATGCCAGCCTGGGTGCTGTGGTCTTC 2324 CGTWDASLGAGVF 1941 TGCGGAACATGGGATGCCAGCCTGGGTGCGGGGGTCTTC 2325 CGTWDARLSGLYVF 1942 TGCGGAACATGGGATGCTAGGCTGAGTGGCCTTTATGTCTTC 2326 CGTWDARLGGAVF 1943 TGTGGAACCTGGGATGCGAGACTGGGTGGTGCAGTCTTC 2327 CGTWDANLRAGVF 1944 TGCGGAACATGGGATGCCAATCTGCGTGCTGGGGTCTTC 2328 CGTWDAIISGWVF 1945 TGCGGAACATGGGATGCTATCATAAGTGGTTGGGTGTTC 2329 CGTWDAGQSVWVF 1946 TGCGGAACATGGGATGCCGGCCAGAGTGTTTGGGTGTTC 2330 CGTWDAGLTGLYVF 1947 TGCGGCACATGGGATGCCGGGCTGACTGGCCTTTATGTCTTC 2331 CGTWDAGLSVYVF 1948 TGCGGAACTTGGGATGCCGGTCTGAGTGTTTATGTCTTC 2332 CGTWDAGLSTGVF 1949 TGCGGGACATGGGATGCCGGCCTGAGTACTGGGGTCTTC 2333 CGTWDAGLSGDVF 1950 TGCGGAACATGGGATGCCGGCCTGAGTGGGGACGTTTTC 2334 CGTWDAGLSAGYVF 1951 TGCGGAACATGGGATGCCGGCCTGAGTGCTGGTTATGTCTTC 2335 CGTWDAGLRVWVF 1952 TGCGGAACATGGGATGCCGGCCTGCGTGTTTGGGTGTTC 2336 CGTWDAGLREIF 1953 TGCGGAACATGGGATGCCGGCCTGAGGGAAATTTTC 2337 CGTWASSLSSWVF 1954 TGCGGAACATGGGCCAGCAGCCTGAGTTCTTGGGTGTTC 2338 CGTWAGSLSGHVF 1955 TGCGGAACATGGGCTGGCAGCCTGAGTGGTCATGTCTTC 2339 CGTWAGSLSAAWVF 1956 TGCGGAACATGGGCTGGCAGCCTGAGTGCCGCTTGGGTGTTC 2340 CGTWAGSLNVYWVF 1957 TGCGGAACATGGGCTGGCAGCCTGAATGTTTATTGGGTGTTC 2341 CGTWAGNLRPNWVF 1958 TGCGGAACATGGGCTGGCAACCTGAGACCTAATTGGGTGTTC 2342 CGTRGSLGGAVF 1959 TGCGGAACAAGGGGTAGCCTGGGTGGTGCGGTGTTC 2343 CGTRDTTLSVPVF 1960 TGCGGAACAAGGGATACCACCCTGAGTGTCCCGGTGTTC 2344 CGTRDTSLNIEIF 1961 TGCGGAACACGGGATACCAGCCTCAATATTGAAATCTTC 2345 CGTRDTSLNDVF 1962 TGTGGAACACGGGATACCAGCCTGAATGATGTCTTC 2346 CGTRDTRLSIVVF 1963 TGCGGAACACGGGATACCCGCCTGAGTATTGTGGTTTTC 2347 CGTRDTILSAEVF 1964 TGCGGCACACGGGATACCATCCTGAGTGCTGAGGTGTTC 2348 CGTRDRSLSGWVF 1965 TGCGGAACACGGGATAGAAGCCTGAGTGGTTGGGTGTTC 2349 CGSWYYNVFLF 1966 TGCGGATCATGGTATTACAATGTCTTCCTTTTC 2350 CGSWHSSLNLVVF 1967 TGCGGATCTTGGCATAGCAGCCTCAACCTTGTCGTCTTC 2351 CGSWGSGLSAPYVF 1968 TGCGGATCATGGGGTAGTGGCCTGAGTGCCCCTTATGTCTTC 2352 CGSWESGLGAWLF 1969 TGCGGTTCGTGGGAAAGCGGCCTGGGTGCTTGGCTGTTC 2353 CGSWDYGLLLF 1970 TGCGGATCCTGGGATTACGGCCTCCTACTCTTC 2354 CGSWDVSLTAVF 1971 TGCGGTTCATGGGATGTCAGCCTGACTGCTGTTTTC 2355 CGSWDVSLNVGIF 1972 TGCGGATCCTGGGATGTCAGTCTCAATGTTGGCATTTTC 2356 CGSWDTTLRAWVF 1973 TGCGGATCATGGGATACCACCCTGCGTGCTTGGGTGTTC 2357 CGSWDTSPVRAWVF 1974 TGCGGCTCGTGGGATACCAGCCCTGTCCGTGCTTGGGTGTTC 2358 CGSWDTSLSVWVF 1975 TGCGGATCATGGGATACCAGCCTGAGTGTTTGGGTGTTC 2359 CGSWDTSLSAEVF 1976 TGCGGATCATGGGATACCAGCCTGAGTGCTGAGGTGTTC 2360 CGSWDTSLRAWVF 1977 TGCGGCTCGTGGGATACCAGCCTGCGTGCTTGGGTGTTC 2361 CGSWDTSLRAWAF 1978 TGCGGCTCGTGGGATACCAGCCTGCGTGCTTGGGCGTTC 2362 CGSWDTSLDARLF 1979 TGCGGATCATGGGATACCAGCCTGGATGCTAGGCTGTTC 2363 CGSWDTILLVYVF 1980 TGCGGATCATGGGATACCATCCTGCTTGTCTATGTCTTC 2364 CGSWDRWQAAVF 1981 TGCGGATCATGGGATCGCTGGCAGGCTGCTGTCTTC 2365 CGSWDRSLSGYVF 1982 TGCGGATCATGGGATAGGAGCCTGAGTGGGTATGTCTTC 2366 CGSWDRSLSAYVF 1983 TGCGGATCATGGGATAGAAGCCTGAGTGCTTATGTCTTC 2367 CGSWDRSLSAVVF 1984 TGCGGATCATGGGATAGGAGCCTGAGTGCCGTGGTTTTC 2368 CGSWDNTLGVVLF 1985 TGCGGATCATGGGATAACACCTTGGGTGTTGTTCTCTTC 2369 CGSWDNRLSTVIF 1986 TGCGGATCGTGGGATAACAGACTAAGTACTGTCATCTTC 2370 CGSWDNRLNTVIF 1987 TGCGGAAGCTGGGATAATCGATTGAACACTGTGATTTTC 2371 CGSWDLSPVRVLVF 1988 TGCGGTTCATGGGATCTCAGCCCTGTACGTGTCCTTGTGTTC 2372 CGSWDLSLSAVVF 1989 TGCGGATCATGGGATCTCAGCCTGAGTGCTGTCGTTTTC 2373 CGSWDKNLRAVLF 1990 TGCGGATCATGGGATAAAAACCTGCGTGCTGTGCTGTTC 2374 CGSWDISLSAGVF 1991 TGCGGCTCATGGGATATCAGCCTGAGTGCTGGGGTGTTC 2375 CGSWDIRLSAEVF 1992 TGCGGATCATGGGATATCAGACTGAGTGCAGAGGTCTTC 2376 CGSWDIKLNIGVF 1993 TGCGGATCATGGGACATCAAACTGAATATTGGGGTATTC 2377 CGSWDFSLNYFVF 1994 TGCGGATCATGGGATTTCAGTCTCAATTATTTTGTCTTC 2378 CGSWDASLSTEVF 1995 TGCGGATCATGGGATGCCAGCCTGAGTACTGAGGTGTTC 2379 CGSWDAGLRGWVF 1996 TGCGGATCCTGGGATGCCGGCCTGCGTGGCTGGGTTTTC 2380 CGRWESSLGAVVF 1997 TGCGGAAGATGGGAGAGCAGCCTGGGTGCTGTGGTTTTC 2381 CGRWDFSLSAYVF 1998 TGCGGAAGATGGGATTTTAGTCTGAGTGCTTATGTCTTC 2382 CGQWDNDLSVWVF 1999 TGCGGACAATGGGATAACGACCTGAGTGTTTGGGTGTTC 2383 CGPWHSSVTSGHVL 2000 TGCGGACCCTGGCATAGCAGCGTGACTAGTGGCCACGTGCTC 2384 CGLWDASLSAPTWVF 2001 TGCGGATTATGGGATGCCAGCCTGAGTGCTCCTACTTGGGTGTTC 2385 CGIWHTSLSAWVF 2002 TGTGGAATATGGCACACTAGCCTGAGTGCTTGGGTGTTC 2386 CGIWDYSLDTWVF 2003 TGCGGAATATGGGATTACAGCCTGGATACTTGGGTGTTC 2387 CGIWDTSLSAWVF 2004 TGCGGCATATGGGATACCAGCCTGAGTGCTTGGGTGTTC 2388 CGIWDTRLSVYVF 2005 TGCGGAATTTGGGATACCAGGCTGAGTGTTTATGTCTTC 2389 CGIWDTRLSVYIF 2006 TGCGGAATTTGGGATACCAGGCTGAGTGTTTATATCTTC 2390 CGIWDTNLGYLF 2007 TGTGGAATATGGGATACGAATCTGGGTTATCTCTTC 2391 CGIWDTGLSAVVF 2008 TGCGGTATATGGGATACCGGCCTGAGTGCTGTGGTATTC 2392 CGIWDRSLSAWVF 2009 TGCGGAATATGGGATCGCAGCCTGAGTGCTTGGGTGTTT 2393 CGIRDTRLSVYVF 2010 TGCGGAATTCGGGATACCAGGCTGAGTGTTTATGTCTTC 2394 CGGWSSRLGVGPVF 2011 TGCGGAGGATGGAGTAGCAGACTGGGTGTTGGCCCAGTGTTT 2395 CGGWGSGLSAWVF 2012 TGCGGAGGATGGGGTAGCGGCCTGAGTGCTTGGGTGTTC 2396 CGGWDTSLSAWVF 2013 TGCGGAGGATGGGATACCAGCCTGAGTGCTTGGGTGTTC 2397 CGGWDRGLDAWVF 2014 TGCGGAGGATGGGATAGGGGCCTGGATGCTTGGGTTTTC 2398 CGAWRNNVWVF 2015 TGCGGAGCATGGCGTAATAACGTGTGGGTGTTC 2399 CGAWNRRLNPHSHWVF 2016 TGCGGAGCATGGAACAGGCGCCTGAATCCTCATTCTCATTGGGTG TTC 2400 CGAWHNKLSAVF 2017 TGCGGAGCCTGGCACAACAAACTGAGCGCGGTCTTC 2401 CGAWGSSLRASVF 2018 TGCGGAGCATGGGGTAGCAGCCTGAGAGCTAGTGTCTTC 2402 CGAWGSGLSAWVF 2019 TGCGGAGCATGGGGTAGCGGCCTGAGTGCTTGGGTGTTC 2403 CGAWESSLSAPYVF 2020 TGCGGAGCATGGGAAAGTAGCCTGAGTGCCCCTTATGTCTTC 2404 CGAWESSLNVGLI 2021 TGCGGAGCATGGGAGAGCAGCCTCAATGTTGGACTGATC 2405 CGAWESGRSAGVVF 2022 TGCGGAGCATGGGAGAGCGGCCGGAGTGCTGGGGTGGTGTTC 2406 CGAWDYSVSGWVF 2023 TGCGGAGCTTGGGATTACAGTGTGAGTGGTTGGGTGTTC 2407 CGAWDYSLTAGVF 2024 TGCGGAGCATGGGATTACAGCCTGACTGCCGGAGTATTC 2408 CGAWDYRLSAVLF 2025 TGCGGAGCCTGGGATTACAGACTGAGTGCCGTGCTATTC 2409 CGAWDVRLDVGVF 2026 TGCGGAGCGTGGGATGTTCGTCTGGATGTTGGGGTGTTC 1203 CGAWDTYSYVF 2027 TGCGGAGCATGGGATACCTACAGTTATGTCTTC 1204 CGAWDTTLSGVVF 2028 TGCGGAGCATGGGATACGACCCTGAGTGGTGTGGTATTC 1205 CGAWDTTLSAVIF 2029 TGCGGAGCGTGGGATACTACCCTGAGTGCTGTGATATTC 1206 CGAWDTSQGASYVF 2030 TGCGGCGCATGGGATACCAGCCAGGGTGCGTCTTATGTCTTT 1207 CGAWDTSPVRAGVF 2031 TGCGGAGCATGGGATACCAGCCCTGTACGTGCTGGGGTGTTC 1208 CGAWDTSLWLF 2032 TGCGGAGCATGGGATACCAGCCTGTGGCTTTTC 1209 CGAWDTSLTVYVF 2033 TGCGGAGCATGGGATACCAGCCTGACTGTTTATGTCTTC 1210 CGAWDTSLTAGVF 2034 TGCGGAGCATGGGACACCAGTCTGACTGCTGGGGTGTTC 1211 CGAWDTSLSTVVF 2035 TGCGGAGCTTGGGATACCAGCCTGAGTACTGTGGTTTTC 1212 CGAWDTSLSSRYIF 2036 TGCGGAGCATGGGATACCAGCCTGAGTTCTAGATACATATTC 1213 CGAWDTSLSGYVF 2037 TGCGGAGCATGGGATACCAGCCTGAGTGGTTATGTCTTC 1214 CGAWDTSLSGWVF 2038 TGCGGAGCCTGGGATACCAGCCTGAGTGGCTGGGTGTTC 1215 CGAWDTSLSGVLF 2039 TGCGGAGCATGGGATACCAGTCTGAGTGGTGTGCTATTC 1216 CGAWDTSLSGLVF 2040 TGCGGAGCTTGGGATACCAGCTTGAGTGGTCTTGTTTTC 1217 CGAWDTSLSGFVF 2041 TGCGGAGCTTGGGATACCAGCTTGAGTGGTTTTGTTTTC 1218 CGAWDTSLSGEVF 2042 TGCGGAGCATGGGATACCAGCCTGAGTGGTGAGGTCTTT 1219 CGAWDTSLSDFVF 2043 TGCGGAGCTTGGGATACCAGCTTGAGTGATTTTGTTTTC 1220 CGAWDTSLRTAIF 2044 TGCGGAGCATGGGATACCAGCCTGCGAACTGCGATATTC 1221 CGAWDTSLRLF 2045 TGCGGAGCATGGGATACCAGCCTGCGGCTTTTC 1222 CGAWDTSLNVHVF 2046 TGCGGAGCATGGGATACCAGCCTGAATGTTCATGTCTTC 1223 CGAWDTSLNKWVF 2047 TGCGGAGCATGGGATACCAGCCTCAATAAATGGGTGTTC 1224 CGAWDTRLSARLF 2048 TGCGGAGCATGGGATACCCGCCTCAGTGCGCGGCTGTTC 1225 CGAWDTRLRGF1F 2049 TGCGGAGCATGGGATACCAGACTGAGGGGTTTTATTTTC 1226 CGAWDTNLGNVLL 2050 TGCGGAGCATGGGATACTAATTTGGGGAATGTTCTCCTC 1227 CGAWDTNLGKWVF 2051 TGCGGGGCATGGGATACCAACCTGGGTAAATGGGTTTTC 1228 CGAWDTGLEWYVF 2052 TGCGGAGCATGGGATACCGGCCTTGAGTGGTATGTTTTT 1229 CGAWDRTSGLWLF 2053 TGCGGAGCATGGGATAGGACTTCTGGATTGTGGCTTTTC 1230 CGAWDRSLVAGLF 2054 TGCGGAGCGTGGGATCGTAGCCTGGTTGCTGGACTCTTC 1231 CGAWDRSLTVYVF 2055 TGCGGAGCGTGGGATAGAAGCCTGACTGTTTATGTCTTC 1232 CGAWDRSLSGYVF 2056 TGCGGAGCATGGGATAGAAGCCTGAGTGGTTATGTCTTC 1233 CGAWDRSLSAYVF 2057 TGCGGAGCATGGGATAGAAGCCTGAGTGCTTATGTCTTC 1234 CGAWDRSLSAVVF 2058 TGCGGAGCATGGGATAGAAGCCTGAGTGCGGTGGTATTC 1235 CGAWDRSLSAGVF 2059 TGCGGAGCATGGGATCGCAGCCTGAGTGCTGGGGTTTTC 1236 CGAWDRSLRIVVF 2060 TGCGGAGCGTGGGATCGCAGCCTGCGTATTGTGGTATTC 1237 CGAWDRSLRAYVF 2061 TGCGGAGCATGGGATAGAAGTCTGAGGGCTTACGTCTTC 1238 CGAWDRSLNVWLF 2062 TGCGGAGCATGGGATAGAAGTCTGAATGTTTGGCTGTTC 1239 CGAWDRGLNVGWLF 2063 TGCGGCGCCTGGGATAGGGGCCTGAATGTCGGTTGGCTTTTC 1240 CGAWDNRLSILAF 2064 TGCGGCGCATGGGATAATAGACTGAGTATTTTGGCCTTC 1241 CGAWDNDLTAYVF 2065 TGCGGAGCTTGGGATAATGACCTGACAGCTTATGTCTTC 1242 CGAWDFSLTPLF 2066 TGCGGGGCATGGGATTTCAGCCTGACTCCTCTCTTC 1243 CGAWDDYRGVSIYVF 2067 TGCGGAGCCTGGGATGACTATCGGGGTGTGAGTATTTATGTCTTC 1244 CGAWDDRPSSAVVF 2068 TGTGGAGCATGGGATGACCGGCCTTCGAGTGCCGTGGTTTTC 1245 CGAWDDRLTVVVF 2069 TGCGGAGCATGGGATGACAGACTGACTGTCGTTGTTTTC 1246 CGAWDDRLGAVF 2070 TGCGGAGCGTGGGATGACAGGCTGGGTGCTGTGTTC 1247 CGAWDASLNPGRAF 2071 TGCGGAGCGTGGGATGCCAGCCTGAATCCTGGCCGGGCATTC 1248 CGAWDAGLREIF 2072 TGCGGAGCATGGGATGCCGGCCTGAGGGAAATTTTC 1249 CGAWAGSPSPWVF 2073 TGCGGAGCTTGGGCTGGCAGTCCGAGTCCTTGGGTTTTC 1250 CGAFDTTLSAGVF 2074 TGCGGAGCATTCGACACCACCCTGAGTGCTGGCGTTTTC 1251 CETWESSLSVGVF 2075 TGCGAAACATGGGAGAGCAGCCTGAGTGTTGGGGTCTTC 1252 CETWESSLRVWVF 2076 TGCGAAACATGGGAAAGCAGCCTGAGGGTTTGGGTGTTC 1253 CETWDTSLSGGVF 2077 TGCGAAACGTGGGATACCAGCCTGAGTGGTGGGGTGTTC 1254 CETWDTSLSDFYVF 2078 TGCGAAACATGGGATACCAGCCTGAGTGACTTTTATGTCTTC 1255 CETWDTSLSALF 2079 TGCGAAACATGGGATACCAGCCTGAGTGCCCTCTTC 1256 CETWDTSLRAEVF 2080 TGCGAAACATGGGATACCAGCCTGCGTGCTGAAGTCTTC 1257 CETWDTSLNVVVF 2081 TGCGAAACATGGGATACCAGCCTGAATGTTGTGGTATTC 1258 CETWDTSLGAVVF 2082 TGCGAAACATGGGATACCAGCCTGGGTGCCGTGGTGTTC 1259 CETWDRSLSGVVF 2083 TGCGAAACATGGGATAGAAGCCTGAGTGGTGTGGTATTC 1260 CETWDRSLSAWVF 2084 TGCGAAACATGGGATAGGAGCCTGAGTGCTTGGGTGTTT 1261 CETWDRSLSAVVF 2085 TGCGAAACATGGGATCGCAGCCTGAGTGCTGTGGTCTTC 1262 CETWDRGLSVVVF 2086 TGCGAGACGTGGGATAGAGGCCTGAGTGTTGTGGTTTTC 1263 CETWDRGLSAVVF 2087 TGCGAAACATGGGATAGGGGCCTGAGTGCAGTGGTATTC 1264 CETWDHTLSVVIF 2088 TGCGAAACATGGGATCACACCCTGAGTGTTGTGATATTC 1265 CETWDASLTVVLF 2089 TGCGAAACATGGGATGCCAGCCTGACTGTTGTGTTATTC 1266 CETWDASLSAGVF 2090 TGCGAAACATGGGATGCCAGCCTGAGTGCTGGGGTGTTC 1267 CETWDAGLSEVVF 2091 TGCGAAACGTGGGATGCCGGCCTGAGTGAGGTGGTGTTC 1268 CE1FDTSLSVVVF 2092 TGCGAAACATTTGATACCAGCCTGAGTGTTGTAGTCTTC 1269 CE1FDTSLNIVVF 2093 TGCGAAACATTTGATACCAGCCTAAATATTGTAGTCTTT 1270 CESWDRSRIGVVF 2094 TGCGAATCATGGGATAGAAGCCGGATTGGTGTGGTCTTC 1271 CESWDRSLSARVY 2095 TGCGAAAGTTGGGACAGGAGTCTGAGTGCCCGGGTGTAC 1272 CESWDRSLRAVVF 2096 TGCGAATCCTGGGATAGGAGCCTGCGTGCCGTGGTCTTC 1273 CESWDRSLIVVF 2097 TGCGAATCTTGGGATCGTAGTTTGATTGTGGTGTTC 1274 CESWDNNLNEVVF 2098 TGCGAAAGTTGGGATAACAATTTAAATGAGGTGGTTTTC 1275 CEIWESSPSADDLVF 2099 TGCGAAATATGGGAGAGCAGCCCGAGTGCTGACGATTTGGTGTTC 1276 CEAWDTSLSGAVF 2100 TGCGAAGCATGGGATACCAGCCTGAGTGGTGCGGTGTTC 1277 CEAWDTSLSAGVF 2101 TGCGAAGCATGGGATACCAGCCTGAGTGCCGGGGTGTTC 1278 CEAWDTSLGGGVF 2102 TGCGAAGCATGGGATACCAGCCTGGGTGGTGGGGTGTTC 1279 CEAWDRSLTGSLF 2103 TGCGAAGCATGGGATCGCAGCCTGACTGGTAGCCTGTTC 1280 CEAWDRGLSAVVF 2104 TGCGAAGCGTGGGATAGGGGCCTGAGTGCAGTGGTATTC 1281 CEAWDNILSTVVF 2105 TGCGAAGCCTGGGATAACATCCTGAGTACTGTGGTGTTC 1282 CEAWDISLSAGVF 2106 TGCGAAGCATGGGACATCAGCCTGAGTGCTGGGGTGTTC 1283 CEAWDADLSGAVF 2107 TGCGAAGCATGGGATGCCGACCTGAGTGGTGCGGTGTTC 1284 CATWTGSFRTGHYVF 2108 TGCGCAACATGGACTGGTAGTTTCAGAACTGGCCATTATGTCTTC 1285 CATWSSSPRGWVF 2109 TGCGCAACATGGAGTAGCAGTCCCAGGGGGTGGGTGTTC 1286 CATWHYSLSAGRVF 2110 TGCGCAACATGGCATTACAGCCTGAGTGCTGGCCGAGTGTTC 1287 CATWHTSLSIVQF 2111 TGCGCAACATGGCATACCAGCCTGAGTATTGTGCAGTTC 1288 CATWHSTLSADVLF 2112 TGCGCAACATGGCATAGCACCCTGAGTGCTGATGTGCTTTTC 1289 CATWHSSLSAGRLF 2113 TGCGCAACATGGCATAGCAGCCTGAGTGCTGGCCGACTCTTC 1290 CATWHIARSAWVF 2114 TGCGCAACATGGCATATCGCTCGGAGTGCCTGGGTGTTC 1291 CATWGSSQSAVVF 2115 TGCGCAACATGGGGTAGTAGTCAGAGTGCCGTGGTATTC 1292 CATWGSSLSAGGVF 2116 TGCGCAACATGGGGTAGCAGCCTGAGTGCTGGGGGTGTTTTC 1293 CATWEYSLSVVLF 2117 TGTGCAACATGGGAATACAGCCTGAGTGTTGTGCTGTTC 1294 CATWETTRRASFVF 2118 TGCGCAACATGGGAGACCACCCGACGTGCCTCTTTTGTCTTC 1295 CATWETSLNVYVF 2119 TGCGCAACATGGGAGACCAGCCTGAATGTTTATGTCTTC 1296 CATWETSLNVVVF 2120 TGCGCAACATGGGAAACTAGCCTGAATGTTGTGGTCTTC 1297 CATWETSLNLYVF 2121 TGCGCAACATGGGAGACCAGCCTGAATCTTTATGTCTTC 1298 CATWETGLSAGEVF 2122 TGCGCAACATGGGAGACTGGCCTAAGTGCTGGAGAGGTGTTC 1299 CATWESTLSVVVF 2123 TGCGCGACGTGGGAGAGTACCCTAAGTGTTGTGGTTTTC 1300 CATWESSLSIFVF 2124 TGCGCAACGTGGGAGAGCAGCCTGAGTATTTTTGTCTTC 1301 CATWESSLNTFYVF 2125 TGCGCAACATGGGAAAGCAGCCTCAACACTTTTTATGTCTTC 1302 CATWESRVDTRGLLF 2126 TGCGCAACATGGGAGAGTAGGGTGGATACTCGAGGGTTGTTATTC 1303 CATWESGLSGAGVF 2127 TGCGCAACATGGGAGAGCGGCCTGAGTGGTGCGGGGGTGTTC 1304 CATWEGSLNTFYVF 2128 TGCGCAACATGGGAAGGCAGCCTCAACACTTTTTATGTCTTC 1305 CATWDYSLSAVVF 2129 TGCGCAACTTGGGATTATAGCCTGAGTGCTGTGGTGTTC 1306 CATWDYRLSIVVF 2130 TGCGCAACATGGGATTACAGACTGAGTATTGTGGTATTC 1307 CATWDYNLGAAVF 2131 TGCGCAACATGGGATTATAACCTGGGAGCTGCGGTGTTC 1308 CATWDVTLGVLHF 2132 TGCGCCACATGGGATGTCACCCTGGGTGTCTTGCATTTC 1309 CATWDTTLSVWVF 2133 TGCGCAACATGGGATACAACACTGAGTGTCTGGGTCTTC 1310 CATWDTTLSVVLF 2134 TGCGCAACATGGGATACCACCCTGAGTGTAGTACTTTTC 1311 CATWDTTLSVEVF 2135 TGCGCAACATGGGATACCACCCTGAGTGTTGAGGTCTTC 1312 CATWDTSPSLSGFWVF 2136 TGCGCAACATGGGATACCAGCCCCAGCCTGAGTGGTTTTTGGGTG TTC 1313 CATWDTSLTGVVF 2137 TGCGCAACATGGGATACCAGCCTGACTGGTGTGGTATTC 1314 CATWDTSLTGAVF 2138 TGCGCAACATGGGATACCAGCCTGACTGGTGCGGTGTTC 1315 CATWDTSLTAWVF 2139 TGCGCAACATGGGATACCAGCCTGACTGCCTGGGTATTC 1316 CATWDTSLTAVVF 2140 TGCGCAACATGGGATACCAGCCTGACTGCTGTGGTTTTC 1317 CATWDTSLTAKVF 2141 TGCGCAACATGGGATACTAGCCTGACTGCTAAGGTGTTC 1318 CATWDTSLSVVVF 2142 TGCGCAACATGGGACACCAGCCTGAGTGTTGTGGTTTTC 1319 CATWDTSLSVGVF 2143 TGCGCTACTTGGGATACCAGCCTGAGTGTTGGGGTATTT 1320 CATWDTSLSSWVF 2144 TGCGCAACATGGGATACCAGCCTGAGTTCTTGGGTGTTC 1321 CATWDTSLSGGVL 2145 TGCGCAACATGGGATACCAGCCTGAGTGGTGGGGTACTC 1322 CATWDTSLSGGVF 2146 TGCGCAACATGGGATACCAGCCTGAGTGGTGGGGTGTTC 1323 CATWDTSLSGGRVF 2147 TGCGCAACATGGGATACCAGCCTGAGTGGTGGCCGAGTGTTC 1324 CATWDTSLSGDRVF 2148 TGCGCAACATGGGATACCAGCCTGAGTGGTGACCGAGTGTTC 1325 CATWDTSLSEGVF 2149 TGCGCAACGTGGGATACTAGCCTGAGTGAAGGGGTGTTC 1326 CATWDTSLSAVVL 2150 TGCGCAACCTGGGATACCAGCCTGAGTGCCGTGGTGCTC 1327 CATWDTSLSAVF 2151 TGCGCAACATGGGATACCAGCCTGAGTGCTGTCTTC 1328 CATWDTSLSARVF 2152 TGCGCGACATGGGATACCAGCCTGAGTGCTCGGGTGTTC 1329 CATWDTSLSALF 2153 TGCGCAACATGGGATACCAGCCTGAGTGCCTTATTC 1330 CATWDTSLSAHVF 2154 TGCGCAACATGGGATACCAGCCTGAGTGCTCATGTCTTC 1331 CATWDTSLSAGRVF 2155 TGCGCAACATGGGATACCAGCCTGAGTGCTGGCCGGGTGTTC 1332 CATWDTSLSAEVF 2156 TGCGCAACATGGGATACCAGCCTGAGTGCGGAGGTCTTC 1333 CATWDTSLSADAGGGV 2157 TGCGCAACATGGGATACCAGCCTGAGTGCTGATGCTGGTGGGGGG F GTCTTC 1334 CATWDTSLRVVVF 2158 TGCGCAACATGGGATACCAGCCTGCGTGTCGTGGTATTC 1335 CATWDTSLRGVF 2159 TGCGCAACATGGGATACCAGCCTGAGAGGGGTGTTC 1336 CATWDTSLPAWVF 2160 TGCGCAACATGGGATACCAGCCTGCCTGCGTGGGTGTTC 1337 CATWDTSLNVGVF 2161 TGTGCAACATGGGATACCAGCCTGAATGTTGGGGTATTC 1338 CATWDTSLGIVLF 2162 TGCGCAACATGGGATACCAGCCTGGGTATTGTGTTATTT 1339 CATWDTSLGARVVF 2163 TGCGCAACATGGGACACCAGCCTGGGTGCGCGTGTGGTCTTC 1340 CATWDTSLGALF 2164 TGTGCAACGTGGGATACCAGTCTAGGTGCCTTGTTC 1341 CATWDTSLATGLF 2165 TGCGCAACATGGGATACCAGCCTGGCGACTGGACTGTTC 1342 CATWDTSLAAWVF 2166 TGCGCAACATGGGATACCAGCCTGGCTGCCTGGGTATTC 1343 CATWDTRLSAVVF 2167 TGCGCAACCTGGGATACCAGGCTGAGTGCTGTGGTCTTC 1344 CATWDTRLSAGVF 2168 TGCGCAACATGGGATACCAGGCTGAGTGCTGGGGTGTTC 1345 CATWDTRLLITVF 2169 TGTGCAACGTGGGACACACGTCTACTTATTACGGITTTC 1346 CATWDTLLSVELF 2170 TGCGCAACATGGGACACCCTCCTGAGTGTTGAACTCTTC 1347 CATWDTGRNPHVVF 2171 TGCGCAACATGGGATACTGGCCGCAATCCTCATGTGGTCTTC 1348 CATWDTGLSSVLF 2172 TGCGCAACATGGGATACCGGCCTGTCTTCGGTGTTGTTC 1349 CATWDTGLSAVF 2173 TGCGCAACGTGGGATACCGGCCTGAGTGCGGTTTTC 1350 CATWDRTLSIGVF 2174 TGCGCTACGTGGGATAGGACCCTGAGTATTGGAGTCTTC 1351 CATWDRSVTAVLF 2175 TGCGCAACGTGGGATCGCAGTGTGACTGCTGTGCTCTTC 1352 CATWDRSLSGVVF 2176 TGCGCAACCTGGGATAGGAGCCTGAGTGGTGTGGTGTTC 1353 CATWDRSLSAVVF 2177 TGCGCAACATGGGATAGAAGCCTGAGTGCTGTGGTCTTC 1354 CATWDRSLSAVPWVF 2178 TGCGCAACATGGGATAGAAGCCTGAGTGCTGTTCCTTGGGTGTTC 1355 CATWDRSLSAGVF 2179 TGCGCAACATGGGATCGCAGCCTGAGTGCTGGGGTGTTC 1356 CATWDRSLRAGVF 2180 TGCGCAACGTGGGATAGGAGCCTGCGTGCTGGGGTGTTC 1357 CATWDRSLNVYVL 2181 TGCGCAACATGGGATCGCAGTCTGAATGTTTATGTCCTC 1358 CATWDRILSAEVF 2182 TGCGCAACGTGGGATCGCATCCTGAGCGCTGAGGTGTTC 1359 CATWDRGLSTGVF 2183 TGCGCAACGTGGGATAGAGGCCTGAGTACTGGGGTGTTC 1360 CATWDNYLGAAVF 2184 TGCGCAACATGGGATAACTACCTGGGTGCTGCCGTGTTC 1361 CATWDNTPSNIVVF 2185 TGCGCAACATGGGATAACACGCCTTCGAATATTGTGGTATTC 1362 CATWDNTLSVWVF 2186 TGCGCAACATGGGATAATACACTGAGTGTGTGGGTCTTC 1363 CATWDNTLSVNWVF 2187 TGCGCAACATGGGATAACACCCTGAGTGTCAATTGGGTGTTC 1364 CATWDNTLNVFYVF 2188 TGCGCAACCTGGGATAACACACTGAATGTCTTTTATGTTTTC 1365 CATWDNRLSSVVF 2189 TGTGCGACATGGGATAATCGGCTCAGTTCTGTGGTCTTC 1366 CATWDNRLSAGVL 2190 TGCGCAACATGGGATAACCGCCTGAGTGCTGGGGTGCTC 1367 CATWDNRLSAGVF 2191 TGCGCAACGTGGGATAACAGGCTGAGTGCTGGGGTGTTC 1368 CATWDNRDWVF 2192 TGCGCAACATGGGATAACAGGGATTGGGTCTTC 1369 CATWDNNLGAGVF 2193 TGCGCAACATGGGATAACAACCTGGGTGCTGGGGTGTTC 1370 CATWDNKLTSGVF 2194 TGCGCAACATGGGATAACAAGCTGACTTCTGGGGTCTTC 1371 CATWDNILSAWVF 2195 TGCGCAACATGGGATAACATCCTGAGTGCCTGGGTGTTT 1372 CATWDNDIHSGLF 2196 TGCGCAACCTGGGACAACGATATACATTCTGGGCTGTTC 1373 CATWDLSLSALF 2197 TGCGCAACTTGGGATCTCAGCCTGAGTGCCCTGTTC 1374 CATWDITLSAEVF 2198 TGCGCAACATGGGATATCACCCTGAGTGCTGAGGTGTTC 1375 CATWDISPSAGGVF 2199 TGCGCAACGTGGGATATCAGCCCGAGTGCTGGCGGGGTGTTC 1376 CATWDISLSTGRAVF 2200 TGCGCAACATGGGATATCAGTCTAAGTACTGGCCGGGCTGTGTTC 1377 CATWDISLSQVF 2201 TGCGCAACATGGGATATCAGTCTGAGTCAGGTATTC 1378 CATWDIRLSSGVF 2202 TGCGCAACATGGGATATCAGGCTGAGTAGTGGAGTGTTC 1379 CATWDIGPSAGGVF 2203 TGCGCAACGTGGGATATCGGCCCGAGTGCTGGCGGGGTGTTC 1380 CATWDHSRAGVLF 2204 TGCGCAACATGGGATCACAGCCGGGCTGGTGTGCTATTC 1381 CATWDHSPSVGEVF 2205 TGCGCAACATGGGATCACAGTCCGAGTGTTGGAGAAGTCTTC 1382 CATWDHSLRVGVF 2206 TGCGCAACATGGGATCACAGCCTGCGTGTTGGGGTGTTC 1383 CATWDHSLNIGVF 2207 TGCGCAACATGGGATCACAGCCTGAACATTGGGGTGTTC 1384 CATWDHSLGLWAF 2208 TGCGCAACATGGGATCACAGCCTGGGTCTTTGGGCATTC 1385 CATWDHNLRLVF 2209 TGCGCCACATGGGATCACAATCTGCGTCTTGTTTTC 1386 CATWDHILASGVF 2210 TGCGCGACTTGGGATCACATCCTGGCTTCTGGGGTGTTC 1387 CATWDFSLSVWVF 2211 TGCGCAACATGGGATTTCAGCCTGAGTGTTTGGGTGTTC 1388 CATWDFSLSAWVF 2212 TGCGCAACATGGGATTTCAGCCTGAGTGCTTGGGTGTTC 1389 CATWDDTLTAGVF 2213 TGCGCAACATGGGATGACACCCTCACTGCTGGTGTGTTC 1390 CATWDDRLSAVLF 2214 TGCGCAACATGGGACGACAGGCTGAGTGCTGTGCTTTTC 1391 CATWDDRLDAAVF 2215 TGCGCAACATGGGATGACAGGCTGGATGCTGCGGTGTTC 1392 CATWDATLNTGVF 2216 TGCGCAACATGGGATGCGACCCTGAATACTGGGGTGTTC 1393 CATWDASLSVWLL 2217 TGCGCAACATGGGATGCCAGCCTGAGTGTTTGGCTGCTC 1394 CATWDASLSGGVF 2218 TGCGCGACATGGGATGCCAGCCTGAGTGGTGGGGTGTTC 1395 CATRDTTLSAVLF 2219 TGCGCAACACGGGATACCACCCTCAGCGCCGTTCTGTTC 1396 CATLGSSLSLWVF 2220 TGCGCTACATTGGGTAGTAGCCTGAGTCTCTGGGTGTTC 1397 CATIETSLPAWVF 2221 TGCGCAACAATCGAAACTAGCCTGCCTGCCTGGGTATTC 1398 CATGDRSLTVEVF 2222 TGCGCAACAGGGGACAGAAGCCTGACTGTTGAGGTATTC 1399 CATGDLGLTIVF 2223 TGCGCTACAGGGGATCTCGGCCTGACCATAGTCTTC 1400 CASWDYRGRSGWVF 2224 TGCGCATCATGGGATTACAGGGGGAGATCTGGTTGGGTGTTC 1401 CASWDTTLNVGVF 2225 TGCGCATCATGGGATACCACCCTGAATGTTGGGGTGTTC 1402 CASWDTTLGFVLF 2226 TGCGCTTCATGGGATACCACCCTGGGTTTTGTGTTATTC 1403 CASWDTSLSGGYVF 2227 TGCGCATCATGGGATACCAGCCTGAGTGGTGGTTATGTCTTC 1404 CASWDTSLRAGVF 2228 TGCGCATCATGGGATACCAGCCTCCGTGCTGGGGTGTTC 1405 CASWDTSLGAGVF 2229 TGCGCATCATGGGATACCAGCCTGGGTGCTGGGGTGTTC 1406 CASWDRGLSAVVF 2230 TGCGCATCATGGGACAGAGGCCTGAGTGCAGTGGTGTTC 1407 CASWDNVLRGVVF 2231 TGTGCTAGTTGGGATAACGTCCTGCGTGGTGTGGTATTC 1408 CASWDNRLTAVVF 2232 TGCGCGTCATGGGATAACAGGCTGACTGCCGTGGTTTTC 1409 CASWDASLSVAF 2233 TGCGCATCATGGGATGCAAGCCTGTCCGTCGCTTTC 1410 CASWDAGLSSYVF 2234 TGCGCTTCGTGGGATGCCGGCCTGAGTTCTTATGTCTTC 1411 CASGDTSLSGVIF 2235 TGCGCATCCGGGGATACCAGCCTGAGTGGTGTGATATTC 1412 CARWHTSLSIWVF 2236 TGCGCAAGATGGCATACGAGCCTAAGTATTTGGGTCTTC 1413 CAIWDTGLSPGQVAF 2237 TGCGCAATATGGGATACCGGCCTGAGTCCTGGCCAAGTTGCCTTC 1414 CAAWHSGLGLPVF 2238 TGCGCAGCATGGCATAGCGGCCTGGGTCTCCCGGTCTTC 1415 CAAWDYSLSAGVF 2239 TGCGCAGCATGGGATTACAGCCTGAGTGCTGGGGTGTTC 1416 CAAWDTTLRVRLF 2240 TGCGCAGCCTGGGATACTACCCTGCGTGTTAGGCTGTTC 1417 CAAWDTSLTAWVF 2241 TGCGCAGCATGGGATACCAGCCTGACTGCCTGGGTTTTC 1418 CAAWDTSLSGGVF 2242 TGCGCAGCATGGGATACCAGCTTGAGTGGTGGGGTGTTC 1419 CAAWDTSLSGEAVF 2243 TGCGCAGCATGGGATACCAGCCTGAGTGGCGAGGCTGTGTTC 1420 CAAWDTSLSGAVF 2244 TGCGCAGCATGGGATACCAGCTTGAGTGGTGCGGTGTTC 1421 CAAWDTSLSAWVF 2245 TGCGCAGCATGGGATACCAGCCTGAGTGCCTGGGTGTTC 1422 CAAWDTSLSAGVF 2246 TGCGCAGCATGGGATACCAGCCTGAGTGCTGGGGTATTC 1423 CAAWDTSLDTYVF 2247 TGCGCAGCATGGGATACCAGCCTGGATACTTATGTCTTC 1424 CAAWDTRLSGVLF 2248 TGCGCTGCATGGGATACCCGTCTGAGTGGTGTGTTATTC 1425 CAAWDTRLSAGVF 2249 TGCGCAGCATGGGATACCAGGCTGAGTGCTGGGGTGTTC 1426 CAAWDRSLSTGVF 2250 TGCGCAGCATGGGATCGCAGTCTGAGTACTGGAGTTTTC 1427 CAAWDIRRSVLF 2251 TGCGCAGCGTGGGATATCCGCCGGTCTGTCCTTTTC 1428 CAAWDHTQRLSF 2252 TGCGCTGCGTGGGATCACACTCAGCGTCTTTCCTTC 1429 CAAWDHSLSAGQVF 2253 TGCGCAGCATGGGATCACAGCCTGAGTGCTGGCCAGGTGTTC 1430 CAAVDTGLKEWVF 2254 TGCGCAGCAGTCGATACTGGTCTGAAAGAATGGGTGTTC

The CDRs were prescreened to contain no amino acid liabilities, cryptic splice sites or nucleotide restriction sites. The CDR variation was observed in at least two individuals and comprises the near-germline space of single, double and triple mutations. The order of assembly is seen in FIG. 8C.

The VH domains that were designed include IGHV1-69 and IGHV3-30. Each of two heavy chain VH domains are assembled with their respective invariant 4 framework elements (FW1, FW2, FW3, FW4) and variable 3 CDR (H1, H2, H3) elements. For IGHV1-69, 417 variants were designed for H1 and 258 variants were designed for H2. For IGHV3-30, 535 variants were designed for H1 and 165 variants were designed for H2. For the CDR H3, the same cassette was used in both IGHV1-69 and IGHV-30 since both designed use an identical FW4, and because the edge of FW3 is also identical for both IGHV1-69 and IGHV3-30. The CDR H3 comprises an N-terminus and C-terminus element that are combinatorially joined to a central middle element to generate 1×10¹⁰ diversity. The N-terminal and middle element overlap with a “GGG” glycine codon. The middle and C-terminal element overlap with a “GGT” glycine codon. The CDR H3 comprises 5 subpools that were assembled separately. The various N-terminus and C-terminus elements comprise sequences as seen in Table 10.

TABLE 10 Sequences for N-terminus and C-terminus elements Element SEQ ID NO Sequence Stem A 2255 CARDLRELECEEWT XXX SRGPCVDPRGVAGSFDVW Stem B 2256 CARDMYYDF XXX EVVPADDAFDIW Stem C 2257 CARDGRGSLPRPKGGP XXX YDSSEDSGGAFDIW Stem D 2258 CARANQHF XXX GYHYYGMDVW Stem E 2259 CAKHMSMQ XXX RADLVGDAFDVW

Example 9. Enrichment for GPCR GLP1R Binding Proteins

Antibodies having CDR-H3 regions with a variant fragments of GPCR binding protein that were generated by methods described herein were panned using cell-based methods to identify variants which are enriched for binding to particular GPCRs, as described in Example 4.

Variants of the GLP C-terminus peptide were identified (listed in Table 11) that when embedded in the CDR-H3 region of an antibody, were repeatedly and selectively enriched for binding to GPCR GLP1R.

TABLE 11 Sequences of GLP1 embedded in CDR-H3 SEQ ID NO Sequence 2260 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2261 CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW 2262 CAKHMSMQDYLVIGEGQAAKEFIAWLVKGGPARADLVGDAFDVW 2263 CAKHMSMQEGAVTGEGQDAKEFIAWLVKGRVRADLVGDAFDVW 2264 WAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2265 CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2266 CARANQHFYEQEGTFTSDVSSYLEGQAAKEFIAWLVKGGIRGYHYYGMDVW 2267 CARANQHFTELHGEGQAAKEFIAWLVKGRGQIDIGYHYYGMDVW 2268 CARANQHFLGAGVSSYLEGQAAKEFIAWLVKGDTTGYHYYGMDVW 2269 CARANQHFLDKGTFTSDVSSYLEGQAAKEFIAWLVKGIYPGYHYYGMDVW 2270 CARANQHFGTLSAGEGQAAKEFIAWLVKGGSQYDSSEDSGGAFDIW 2271 CARANQHFGLHAQGEGQAAKEFIAWLVKGSGTYGYHYYGMDVW 2272 CARANQHFGGKGEGQAAKEFIAWLVKGGGSGAGYHYYGMDVW 2273 CAKQMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2274 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGGPARADLVGDAFDVW 2275 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW 2276 CAKHMSMQDYLVIGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

Example 10. Analysis of GLP1R Binding Protein Variants

Antibodies having CDR-H3 regions with variant fragments of GLP1R binding protein were generated by methods described herein were panned using cell-based methods to identify variants which are enriched for binding to GLP1R, as described in Example 4.

Next generation sequence (NGS) enrichment for variants of the GLP1R peptides was performed (data not shown). Briefly, phage populations were deep-sequenced after each round of selection are deep-sequenced to follow enrichment and identify cross-sample clones. Target specific clones were selected after filtering out CHO background clones from the NGS data. For GLP1R peptides, about 2000 VH and VL pairs were barcoded directly from a bacterial colony and sequenced to identify non-identical clones.

GLP1R-1 variant was analyzed for V gene distribution, J gene distribution, V gene family, and CDR3 counts per length. Frequency of V genes IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV3-53, IGHV3-NL1, IGHV3-d, IGHV1-46, IGHV3-h, IGHV1, IGHV3-38, IGHV3-48, IGHV1-18, IGHV1-3, and IGHV3-15 was determined (data not shown). High frequency of IGHV1-69 and IGHV3-30 were observed. Frequency of J genes IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, mIGHJ, IGHJ2, and IGH1 was also determined (data not shown). High frequency of IGHJ3 and IGHJ6 were observed with less frequency of IGHJ and IGHJ4 observed. Frequency of V genes IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, and IGHV1-8 was determined (data not shown). High frequency of IGHV1-69 and IGHV3-30 was observed. Frequency of J genes IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2, and IGH1 was determined (data not shown). High frequency of IGHJ3 and IGHJ6 was observed with less frequency of IGHJ and IGHJ4 observed.

H accumulation and frequency were determined for GLP1R-1, GLP1R-2, GLP1R-3, GLP1R-4, and GLP1R-5 (data not shown).

Sequence analytics were performed for GLP1R-1, GLP1R-2, GLP1R-3, GLP1R-4, and GLP1R-5 variants (data not shown).

Cell binding was determined for the GLP1R variants. FIGS. 9A-90 show the cell binding data for GLP1R-2 (FIG. 9A), GLP1R-3 (FIG. 9B), GLP1R-8 (FIG. 9C), GLP1R-26 (FIG. 9D), GLP1R-30 (FIG. 9E), GLP1R-56 (FIG. 9F), GLP1R-58 (FIG. 9G), GLP1R-10 (FIG. 9H), GLP1R-25 (FIG. 9I), GLP1R-60 (FIG. 9J), GLP1R-70 (FIG. 9K), GLP1R-72 (FIG. 9L), GLP1R-83 (FIG. 9M), GLP1R-93 (FIG. 9N), and GLP1R-98 (FIG. 9O).

GLP1R-3, GLP1R-8, GLP1R-26, GLP1R-56, GLP1R-58 and GLP1R-10 were then analyzed for allosteric effects on GLP1-7-36 peptide in a cAMP assay. FIGS. 10A-100 show graphs of the GLP1R variants on inhibition of GLP1-7-36 peptide induced cAMP activity. GLP1R-3 (FIG. 10B), GLP1R-8 (FIG. 10C), GLP1R-26 (FIG. 10D), GLP1R-30 (FIG. 10E), GLP1R-56 (FIG. 10F), GLP1R-58 (FIG. 10G), GLP1R-10 (FIG. 10H, right graph), GLP1R-25 (FIG. 10I), and GLP1R-60 (FIG. 10J) show allosteric inhibition of GLP1-7-36 peptide induced cAMP activity. FIG. 10H further shows effects of GLPR-10 on cAMP signal as compared to exendin-4 (FIG. 10H, left graph).

GLP1R variants were tested in a cAMP assay to determine if the variants were antagonists in blocking exendin-4 induced cAMP activity. FIGS. 11A-11G depict cell functional data for GLP1R-2 (FIG. 11A), GLP1R-3 (FIG. 11B), GLP1R-8 (FIG. 11C), GLP1R-26 (FIG. 11D), GLP1R-30 (FIG. 11E), GLP1R-56 (FIG. 11F), and GLP1R-58 (FIG. 11G).

GLP1R-2, GLP1R-3, GLP1R-8, GLP1R-26, GLP1R-30, GLP1R-56, and GLP1R-58 were then analyzed for allosteric effects on exendin-4 in a cAMP assay. FIGS. 12A-12G depict graphs of GLP1R-2 (FIG. 12A), GLP1R-3 (FIG. 12B), GLP1R-8 (FIG. 12C), GLP1R-26 (FIG. 12D), GLP1R-30 (FIG. 12E), GLP1R-56 (FIG. 12F), and GLP1R-58 (FIG. 12G) variants on inhibition of Exendin-4 peptide induced cAMP activity. Table 12 shows the EC50 (nM) data for Exendin-4 alone or with GLP1R-2, GLP1R-3, GLP1R-8, GLP1R-26, GLP1R-30, GLP1R-56, and GLP1R-58.

TABLE 12 EC50 (nM) Data EC50 fold-diff Fxendin-4 alone 0.12 +GLP1R-2 0.12 1.0 +GLP1R-3 0.63 5.4 +GLP1R-8 0.47 4.0 +GLP1R-26 0.77 6.5 +GLP1R-30 0.11 1.0 +GLP1R-56 0.82 7.0 +GLP1R-58 0.27 2.3

FACS screening was performed on GLP1R variants. GLP1R-2, GLP1R-3, GLP1R-8, GLP1R-10, GLP1R-25, GLP1R-26, GLP1R-30, GLP1R-56, GLP1R-58, GLP1R-60, GLP1R-70, GLP1R-72, GLP1R-83, GLP1R-93, and GLP1R-98 were identified as seen in Table 13. GLP1R-3, GLP1R-8, GLP1R-56, GLP1R-58, GLP1R-60, GLP1R-72, and GLP1R-83 comprise the GLP1 motif. See FIG. 13. GLP1R-25, GLP1R-30, GLP1R-70, GLP1R-93, and GLP1R-98 comprise the GLP2 motif. See FIG. 13. GLP1R-50 and GLP1R-71 comprise the CC chemokine 28 motif.

TABLE 13 GLP1R Variants SEQ ID NO Variant Sequence 2277 GLP1R-1 CARANQHFVDLYGWHGVPKGYHYYGMDVW 2278 GLP1R-2 CARDMYYDFETVVEGIQWYEALKAGKLGEVVPADDAFDIW 2279 GLP1R-3 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2280 GLP1R-8 CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW 2281 GLP1R-10 CARANQHFFVPGSLKVWLKGVAPESSSEYDSSEDSGGAFDIW 2282 GLP1R-25 CARANQHFLSHAGAARDFINWLIQTKITGLGSGYHYYGMDVW 2283 GLP1R-26 CAKHMSMQEGVLQGQIPSTIDWEGLLHLIRADLVGDAFDVW 2284 GLP1R-30 CARDMYYDFLKIGDNLAARDFINWLIQTKITDGTDTEVVPADDAFDIW 2285 GLP1R-50 CARDGRGSLPRPKGGPKFVPGKHETYGHKTGYRLRPGYHYYGMDVW 2286 GLP1R-56 CARANQHFFSGAEGEGQAAKEFIAWLVKGIIPGYHYYGMDVW 2287 GLP1R-58 CARANQHFGLHAQGEGQAAKEFIAWLVKGSGTYGYHYYGMDVW 2288 GLP1R-60 CAKHMSMQDYLVIGEGQAAKEFIAWLVKGGPARADLVGDAFDVW 2289 GLP1R-70 CARDGRGSLPRPKGGPPSSGRDFINWLIQTKITDGFRYDSSEDSGGAFDIW 2290 GLP1R-71 CARDLRELECEEWTRHGGKKHHGKRQSNRAHQGKHETYGHKTGSLVPSRGPCVDPR GVAGSFDVW 2291 GLP1R-72 CARDMYYDFHPEGTFTSDVSSYLEGQAAKEFIAWLVKGSLIYEVVPADDAFDIW 2292 GLP1R-80 CARANQHFGPVAGGATPSEEPGSQLTRAELGWDAPPGQESLADELLQLGTEHGYHYY GMDVW 2293 GLP1R-83 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2294 GLP1R-93 CARANQHFLSHAGAARDFINWLIQTKITGLGSGYHYYGMDVW 2295 GLP1R-98 CARDGRGSLPRPKGGPHSGRLGSGYKSYDSSEDSGGAFDIW *bold corresponds to GLP1 or GLP2 motif

The GLP1R variants were assed for aggregation. Size exclusion chromatography (SEC) was performed on GLP1R-30 and GLP1R-56 variants. 82.64% of GLP1R-30 was monomeric (˜150 Kd). 97.4% of GLP1R-56 was monomeric (˜150 Kd).

Example 11. GPCR Binding Protein Functionality

For a GPCR binding protein, the top 100-200 scFvs from phage-selections were converted to full-length immunoglobulins. After immunoglobulin conversion, the clones were transiently transfected in ExpiCHO to produce immunoglobulins. Kingfisher and Hamilton were used for batch IgG purifications followed by lab-chip to collect purity data for all purified immunoglobulins. High yields and purities were obtained from 10 mL cultures as seen in Table 14.

TABLE 14 Immunoglobulin Purity Percentage IgG % Name Purity mAb1 100 mAb2 100 mAb3 100 mAb4 100 mAb5 98 mAb6 100 mAb7 97 mAb8 100 mAb9 100 mAb10 100 mAb11 100 mAb12 100 mAb13 100 mAb14 100 mAb15 100

Stable cell lines expressing GPCR targets were then generated and confirmed by FACS (data not shown). Cells expressing >80% of the target were then directly used for cell-based selections. Five rounds of selections were carried out against cells overexpressing target of interest. 10⁸ cells were used for each round of selection. Before selection on target expressing cells, phage from each round was first depleted on 10⁸ CHO background cells. Stringency of selections was increased by increasing the number of washes in subsequent rounds of selection. Enrichment ratios were monitored for each round of selection.

Purified IgGs were tested for cell-binding affinity using FACS (FIGS. 14A-14C) and cAMP activity (FIG. 14D). Allosteric inhibition was observed.

Purified IgGs were tested using BVP ELISA. BVP ELISA showed some clones comprising BVP scores comparable to comparator antibodies (data not shown).

Example 12. GLP1R scFv Modulators

This example illustrates identification of GLP1R modulators.

Library Panning

The GPCR1.0/2.0 scFv-phage library was incubated with CHO cells for 1 hour at room temperature (RT) to deplete CHO cell binders. After incubation, the cells were pelleted by centrifuging at 1,200 rpm for 10 minutes to remove the non-specific CHO cell binders. The phage supernatant, which has been depleted of CHO cell binders, was then transferred to GLP1R expressing CHO cells. The phage supernatant and GLP1R expressing CHO cells were incubated for 1 hour at RT to select for GLP1R binders. After incubation, the non-binding clones were washed away by washing with PBS several times. Finally, to selectively elute the agonist clones, the phage bound to the GLP1R cells were competed off with 1 μM of the natural ligand of GLP1R, GLP1 peptide (residues 7 to 36). The clones that eluted off the cells were likely binding to the GLP1 ligand binding epitope on GLP1R. Cells were pelleted by centrifuging at 1,200 rpm for 10 minutes to remove clones that were still binding to GLP1R on the cells, and were not binding to the endogenous GLP1 ligand binding site (orthosteric site). The supernatant was amplified in TG1 E. coli cells for the next round of selection. This selection strategy was repeated for five rounds. Amplified phage from a round was used as the input phage for the subsequent round, and the stringency of washes were increased in each subsequent round of selections. After five rounds of selection, 500 clones from each of round 4 and round 5 were Sanger sequenced to identify clones of GLP1R modulators. Seven unique clones were reformatted to IgG2, purified and tested in binding by FACS and functional assays.

Binding Assays

Seven GLP1R scFv clones (GLP1R-238, GLP1R-239, GLP1R-240, GLP1R-241, GLP1R-242, GLP1R-243, and GLP1R-244) and two GLP1R IgGs (pGPCR-GLP1R-43 and pGPCR-GLP1R-44, Janssen Biotech, J&J) used as controls were tested in binding assays coupled to flow cytometry analysis. CDR3 sequences (Table 15), heavy chain sequences (Table 16), and light chain sequences (Table 17) for GLP1R-238, GLP1R-239, GLP1R-240, GLP1R-241, GLP1R-242, GLP1R-243, and GLP1R-244 are seen below.

TABLE 15 CDR3 sequences SEQ ID NO. Variant CDR-H3 Sequence 2296 GLP1R-238 CARANQHFSQAGRAARVPGPSSSLGPRGYHYYGMDVW 2297 GLP1R-239 CAKHMSMQSQGLDNLAARDFINWLIQTKITDGFELSRADLVGDAFDVW 2298 GLP1R-240 CARDMYYDFFGLGTFTSDVSSYLEGQAAKEFIAWLVKGVSPEVVPADDAFDIW 2299 GLP1R-241 CAKHMSMQGSVAGGTFTSDVSSYLEGQAAKEFIAWLVKGGPSFIRADLVGDAFDVW 2300 GLP1R-242 CAKHMSMQADTGTFTSDVSSYLEGQAAKEFIAWLVKGEFSSRADLVGDAFDVW 2301 GLP1R-243 CARANQHFFGKGDNLAARDFINWLIQTKITDGSNPGYHYYGMDVW 2302 GLP1R-244 CARANQHFAATGAGEGQAAKEFIAWLVKGRVEIGYHYYGMDVW *bold correspond to GLP-1 or GLP-2 motif

TABLE 16 Variable Heavy Chain Sequences SEQ ID NO. Variant Variable Heavy Chain Sequence 2303 GLP1R- MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGSFSSHAISWVRQA 238 PGQGLEWMGGIIPIFGAPNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARAN QHFSQAGRAARVPGPSSSLGPRGYHYYGMDVWGQGTLVTVSSASASTKGPSVFPLAPCS RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG 2304 GLP1R- MEWSWVFLFFLSVTTGVHSQVQLVESGGGVVQPGRSLRLSCAASGFDFSNYGMHWVRQ 239 APGKGLEWVADISYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KHMSMQSQGLDNLAARDFINWLIQTKITDGFELSRADLVGDAFDVWGQGTLVTVSSASA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG 2305 GLP1R- MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFNNYGISWVRQ 240 APGQGLEWMGGIIPVFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR DMYYDFFGLGTFTSDVSSYLEGQAAKEFIAWLVKGVSPEVVPADDAFDIWGQGTLVTVS SASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG 2306 GLP1R- MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISWVRQ 241 APGQGLEWMGGIIPIFGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKH MSMQGSVAGGTFTSDVSSYLEGQAAKEFIAWLVKGGPSFIRADLVGDAFDVWGQGTLV TVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2307 GLP1R- MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYEISWVRQA 242 PGQGLEWMGGIIPILGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKHM SMQADTGTFTSDVSSYLEGQAAKEFIAWLVKGEFSSRADLVGDAFDVWGQGTLVTVSS ASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 2308 GLP1R- MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGINWVRQ 243 APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARA NQHFFGKGDNLAARDFINWLIQTKITDGSNPGYHYYGMDVWGQGTLVTVSSASASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVL TVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG 2309 GLP1R- MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA 244 PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARAN QHFAATGAGEGQAAKEFIAWLVKGRVEIGYHYYGMDVWGQGTLVTVSSASASTKGPSV FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVV HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG

TABLE 17 Variable Light Chain Sequences SEQ ID NO. Variant Variable Light Chain Sequence 2310 GLP1R- MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSTSNIANNYVSWYQQL 238 PGTAPKLLIYANNRRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGAWDVRLDVGV FGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLS 2311 GLP1R- MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSTSNIEKNYVSWYQQL 239 PGTAPKLLIYGNDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWENRLSAVV FGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2312 GLP1R- MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSSIGNNYVSWYQQL 240 PGTAPKLLIYANNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWSSSPRGWVF GGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2313 GLP1R- MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGISSNIGNNYVSWYQQL 241 PGTAPKLLIYDDDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDNILSAAVF GGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2314 GLP1R- MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSNIENNDVSWYQQL 242 PGTAPKLLIYGNDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDNTLSAGV FGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2315 GLP1R- MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSRSNIGKNYVSWYQQ 243 LPGTAPKLLIYENNERPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCSSYTTSNTQVFG GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2316 GLP1R- MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNVVSWYQQL 244 PGTAPKLLIYDNDKRRSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGSWDTSLSVWV FGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Briefly, flag-GLP1R-GFP expressing CHO cells (CHO-GLP1R) and CHO-parent cells were incubated with 100 nM IgG for 1 hour on ice, washed three times and incubated with Alexa 647 conjugated goat-anti-human antibody (1:200) for 30 minutes on ice, followed by three washes. All incubations and washes were performed in buffer containing PBS and 0.5% BSA. For titrations, IgG was serially diluted 1:3 starting from 100 nM. Cells were analyzed by flow cytometry and hits in which IgG was found to bind to CHO-GLP1R were identified by measuring the GFP signal against the Alexa 647 signal. GLP1R-238, GLP1R-240, GLP1R-241, GLP1R-242, GLP1R-243, and GLP1R-244 were found to bind to CHO-GLP1R. GLP1R-238 bound equally to CHO-GLP1R and CHO-parent cells and thus appears to be a non-specific binder. Analyses of binding assays with IgG titrations presented as binding curves plotting IgG concentrations against MFI (mean fluorescence intensity) are seen in FIGS. 15A-15H. Flow cytometry data of binding assays presented as dot plots with 100 nM IgG are seen in FIGS. 16A-16I.

Functional Assays

All GLP1R scFv clones, as well as pGPCR-GLP1R-43 and pGPCR-GLP1R-44, were assayed for their potential effects on GLP1R signaling by performing cAMP assays (Eurofins DiscoverX Corporation). These assays involve CHO cells that were engineered to overexpress naturally Gas-coupled wildtype GLP1R and were designed to detect changes in intracellular cAMP levels in response to agonist stimulation of the receptor. The technology involved in detecting cAMP levels was a no wash gain-of-signal competitive immunoassay based on Enzyme Fragment Complementation technology and produced a luminescent signal that was directly proportional to the amount of cAMP in the cells. Experiments were designed to determine agonist or allosteric activity of the IgGs. To test for agonist activity of the IgGs, cells were stimulated with IgGs (titrations 1:3 starting from 100 nM) or with the known agonist GLP1 (7-36) peptide (titrations 1:6 starting from 12.5 nM) for 30 minutes at 37° C. To test for allosteric activity of the IgGs, cells were incubated with IgGs at a fixed concentration of 100 nM for 1 hour at room temperature to allow binding, followed by stimulation with GLP1 (7-36) peptide (titrations 1:6 starting from 12.5 nM) for 30 minutes at 37° C. Intracellular cAMP levels were detected by following the assay kit instructions.

As seen in FIGS. 17A-17B, none of the IgGs initiated an agonist signal. GLP1R-241 was also tested for cAMP allosteric effect (FIG. 17C), beta-arrestin recruitment (FIG. 17D), and internalization (FIG. 17E). Several of the IgGs acted as negative allosteric modulators by changing the signaling response of these cells to GLP1 (7-36) in an inhibitory manner as seen in FIGS. 18A-18B. Table 18 shows the EC50 (nM) values corresponding to FIG. 18A and Table 19 shows the EC50 corresponding to FIG. 18B.

TABLE 18 EC50 (nM) Values + no Ab +GLP1R-238 +GLP1R-239 +GLP1R-240 +GLP1R-241 +GLP1R-242 GLP1R-243 GLP1R-244 EC50 0.05946 0.08793 0.07995 0.06539 0.1027 ~0.06532 0.1282 0.1536

TABLE 19 EC50 (nM) Values pGPCR- pGPCR- +no Ab 43-GLP1R 44-GLP1R EC50 0.05946 2.948 3.485

The data shows pharmacological and functional effects of GLP1R modulators.

Example 13: GLP1R Agonists and Antagonists

This example illustrates identification of GLP1R agonists and antagonists.

Experiments were performed similarly to Example 12. Six GLP1R immunoglobulins (IgGs) were assayed for binding and functional assays to determine which clones were agonists or antagonists. The GLP1R IgGs tested included GLP1R-59-2, GLP1R-59-241, GLP1R-59-243, GLP1R-3, GLP1R-241, and GLP1R-2. GLP1R-241, GLP1R-3, and GLP1R-2 were previously described in Examples 10 and 12. Heavy chain sequences for GLP1R-59-2, GLP1R-59-241, GLP1R-59-243, GLP1R-43-8, and GLP1R-3 is seen in Table 20.

TABLE 20 Variable Heavy Chain Sequences SEQ ID NO. Variant Variable Heavy Chain Sequence 2317 GLP1R-59-2 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAVISYDAGNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDMYYDFETVVEGIQWYEA LKAGKLGEVVPADDAFDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSN TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2318 GLP1R-59- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISWVRQAPGQGLEWMGGIIPIFGTTN 241 YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKHMSMQGSVAGGTFTSDVSSY LEGQAAKEFIAWLVKGGPSFIRADLVGDAFDVWGQGTLVTVSSASASTKGPSVFPLAPCS RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG 2319 GLP1R-59- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGINWVRQAPGQGLEWMGGIIPIFGTAN 243 YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFFGKGDNLAARDFINW LIQTKITDGSNPGYHYYGMDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD HKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG 2320 GLP1R-3 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSFISYDESNKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVTGEGQAAKEF IAWLVKGRVRADLVGDAFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2321 GLP1R-43-8 MEWSWVFLFFLSVTTGVHSEVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQA PGKEREGVAAINNFGTTKYADSAKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAV RWGPHNDDRYDWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG

The GLP1R IgGs were characterized for thermal ramp stability (Tm and Tagg). The UNcle platform was used to characterize the IgGs and the data is seen in Table 21.

TABLE 21 Thermal Ramp Stability Measurements Average Average Average % CV SD Tm1 % CV Tm2 % CV Tagg 266 Tagg Tagg Sample (° C.) Tm1 SD Tm1 (° C.) Tm2 SD Tm2 (° C.) 266 266 GLP1R-59-2 60.6 0.08 0.05 84.6 0.71 0.6 58.3 0.29 0.17 GLP1R-59-241 66 6.52 4.3 73.6 0.41 0.3 57.8 0.69 0.4 GLP1R-59-243 60.9 0.33 0.2 75.2 0.8 0.6 55.9 0.72 0.4 GLP1R-3 66.7 0.6 0.4 73.5 0.54 0.4 68.4 0.58 0.4 GLP1R-241 68.2 0.82 0.56 75.7 0.94 0.71 65.9 0.76 0.5 GLP1R-2 61.8 1.17 0.72 74.8 1.27 0.95 60.5 0.12 0.07

The GLP1R IgGs were then assayed in binding assays coupled to flow cytometry analysis using similar methods as described in Example 12. Briefly, stably expressing Flag-GLP1R-GFP CHO cells or CHO-parent cells were incubated with primary IgG (100 nM or 1:3 titrations). Secondary antibody incubation involved Alexa 647 conjugated goat-anti-human IgG. Flow cytometry measured the GFP signal against the Alexa 647 signal to identify IgGs that specifically bound to the target (GLP1R). Ligand competition assays involved co-incubating the primary IgG with 1 μM GLP1 (7-36). Data for GLP1R-59-2, GLP1R-59-241, GLP1R-59-243, GLP1R-3, GLP1R-241, and GLP1R-2 are seen in FIGS. 19A-19F.

Functional assays were also performed using the GLP1R IgGs using similar methods as described in Example 12. Briefly, cAMP, beta-arrestin recruitment and activated receptor internalization assays were obtained from Eurofins DiscoverX and utilized untagged GLP-1R overexpressing CHO-K1 or U2OS cells. These were used to test for either agonist activity of the IgGs as compared with GLP1 (7-36) or for antagonistic activity of the IgGs by pre-incubating cells with IgGs and examining their effects on GLP1 (7-36)-induced signaling. For the cAMP assays, following GLP1 (7-36) or IgG stimulation, the cellular cAMP levels are measured using a homogenous, no wash, gain-of-signal competitive immunoassay based on Enzyme Fragment Complementation (EFC) technology. Data from the functional assays for GLP1R-59-2, GLP1R-59-241, GLP1R-59-243, GLP1R-3, GLP1R-241, and GLP1R-2 is seen in FIGS. 20A-20F. The EC50 (nM) data for GLP1R-59-2, GLP1R-59-241, GLP1R-59-243, GLP1R-3, GLP1R-241, and GLP1R-2 is seen in Tables 22-23. As seen in Table 23, the EC50 data for GLP1R-3 showed a 2.2-fold difference. The EC50 data for GLP1-241 showed a 1.7-fold difference. The EC50 data for GLP1R-2 showed a 0.8-fold difference.

TABLE 22 EC50 (nM) for GLP1R-59-2, GLP1R-59-241, and GLP1R-59-243 GLP1R IgG EC50 GLP1 (7-36) EC50 GLP1R-59-2 0.842 0.4503 GLP1R-59-241 0.7223 0.4731 GLP1R-59-243 0.8209 0.4731

TABLE 23 EC50 (nM) for GLP1R-3, GLP1R-241, and GLP1R-2 GLP1R IgG (+100 nM) EC50 No Antibody EC50 GLP1R-3 1.311 0.6053 GLP1R-241 0.1027 0.05946 GLP1R-2 0.07947 0.1031

GLP1R-3 was also assayed to determine specificity of GLP1R versus GL1P2R binding and determined to be specific for GLP1R over GLP2R (data not shown). Binding of GLP1R-3, GLP1R-59-242, and GLP1R-43-8 on mouse, macaca, and human GLP1R was determined. GLP1R-3 at 100 nM, GLP1R-59-242 at 100 nM, and GLP1R-43-8 at 100 nM were found to bind mouse, macaca, and human GLP1R (data not shown). GLP1R-3 was also found to bound human pancreatic precursor cells expressing endogenous GLP1R.

Binding of GLP1R-59-2, GLP1R-59-241, and GLP1R-59-243 on mouse, macaca, and human GLP1R was determined. GLP1R-59-2 at 100 nM, GLP1R-59-241 at 100 nM, and GLP1R-59-243 at 50 nM were found to bind mouse, macaca, and human GLP1R (data not shown). GLP1R-59-2 was also found to bound human pancreatic precursor cells expressing endogenous GLP1R.

This example shows GLP1R IgGs with agonistic and antagonist properties. Several of the IgGs induced cAMP signaling, beta-arresting recruitment, and receptor internalization similar to GLP1 (7-36).

Example 14: VHH Libraries

Synthetic VHH libraries were developed. For the ‘VHH Ratio’ library with tailored CDR diversity, 2391 VHH sequences (iCAN database) were aligned using Clustal Omega to determine the consensus at each position and the framework was derived from the consensus at each position. The CDRs of all the 2391 sequences were analyzed for position-specific variation, and this diversity was introduced in the library design. For the ‘VHH Shuffle’ library with shuffled CDR diversity, the iCAN database was scanned for unique CDRs in the nanobody sequences. 1239 unique CDR1's, 1600 unique CDR2's, and 1608 unique CDR3's were identified and the framework was derived from the consensus at each framework position amongst the 2391 sequences in the iCAN database. Each of the unique CDR's was individually synthesized and shuffled in the consensus framework to generate a library with theoretical diversity of 3.2×10{circumflex over ( )}9. The library was then cloned in the phagemid vector using restriction enzyme digest. For the ‘VHH hShuffle’ library (a synthetic “human” VHH library with shuffled CDR diversity), the iCAN database was scanned for unique CDRs in the nanobody sequences. 1239 unique CDR1's, 1600 unique CDR2's, and 1608 unique CDR3's were identified and framework 1, 3, and 4 was derived from the human germline DP-47 framework. Framework 2 was derived from the consensus at each framework position amongst the 2391 sequences in the iCAN database. Each of the unique CDR's was individually synthesized and shuffled in the partially humanized framework using the NUGE tool to generate a library with theoretical diversity of 3.2×10{circumflex over ( )}9. The library was then cloned in the phagemid vector using the NUGE tool.

The Carterra SPR system was used to assess binding affinity and affinity distribution for VHH-Fc variants. VHH-Fc demonstrate a range of affinities for TIGIT, with a low end of 12 nM K_(D) and a high end of 1685 nM K_(D) (data not shown). Table 23A provides specific values for the VHH-Fc clones for ELISA, Protein A (mg/ml), and K_(D) (nM). FIG. 21A and FIG. 21B depict TIGIT affinity distribution for the VHH libraries, over the 20-4000 affinity threshold (FIG. 21A; monovalent K_(D)) and the 20-1000 affinity threshold (FIG. 21B; monovalent K_(D)). Out of the 140 VHH binders tested, 51 variants had affinity <100 nM, and 90 variants had affinity <200 nM.

TABLE 23A ELISA, Protein A, and K_(D) of VHH-Fc Clones ProA Clone ELISA Library (mg/ml) K_(D) (nM) Variant 31-1 5.7 VHH hShuffle 0.29 12 Variant 31-6 9.6 VHH hShuffle 0.29 14 Variant 31-26 5.1 VHH hShuffle 0.31 19 Variant 30-30 8 VHH Shuffle 0.11 23 Variant 31-32 8 VHH hShuffle 0.25 27 Variant 29-10 5 VHH Ratio 0.19 32 Variant 29-7 7.3 VHH Ratio 0.28 41 Variant 30-43 13.5 VHH Shuffle 0.18 44 Variant 31-8 12.7 VHH hShuffle 0.29 45 Variant 31-56 11.7 VHH hShuffle 0.26 46 Variant 30-52 4.2 VHH Shuffle 0.22 49 Variant 31-47 8.8 VHH hShuffle 0.23 53 Variant 30-15 9.3 VHH Shuffle 0.26 55 Variant 30-54 5.5 VHH Shuffle 0.3 58 Variant 30-49 10.3 VHH Shuffle 0.26 62 Variant 29-22 3.4 VHH Ratio 0.27 65 Variant 29-30 9.2 VHH Ratio 0.28 65 Variant 31-35 5.7 VHH hShuffle 0.24 66 Variant 29-1 10.4 VHH Ratio 0.09 68 Variant 29-6 6.8 VHH Ratio 0.29 69 Variant 31-34 6 VHH hShuffle 0.32 70 Variant 29-12 6.2 VHH Ratio 0.23 70 Variant 30-1 5.4 VHH Shuffle 0.39 71 Variant 29-33 3.9 VHH Ratio 0.15 74 Variant 30-20 4.6 VHH Shuffle 0.19 74 Variant 31-20 6.6 VHH hShuffle 0.37 74 Variant 31-24 3.1 VHH hShuffle 0.15 75 Variant 30-14 9.9 VHH Shuffle 0.19 75 Variant 30-53 7.6 VHH Shuffle 0.24 78 Variant 31-39 9.9 VHH hShuffle 0.32 78 Variant 29-18 10.9 VHH Ratio 0.19 78 Variant 30-9 8 VHH Shuffle 0.4 79 Variant 29-34 8.6 VHH Ratio 0.21 80 Variant 29-27 8.6 VHH Ratio 0.18 82 Variant 29-20 5.9 VHH Ratio 0.26 83 Variant 30-55 6 VHH Shuffle 0.41 85 Variant 30-39 6.1 VHH Shuffle 0.07 88 Variant 31-15 6.2 VHH hShuffle 0.32 88 Variant 29-21 4.3 VHH Ratio 0.23 88 Variant 29-37 5.3 VHH Ratio 0.26 89 Variant 29-40 6.6 VHH Ratio 0.31 90 Variant 31-30 3.2 VHH hShuffle 0.33 93 Variant 31-10 12.3 VHH hShuffle 0.31 94 Variant 29-3 13.6 VHH Ratio 0.11 94 Variant 30-57 5.2 VHH Shuffle 0.24 95 Variant 29-31 4.4 VHH Ratio 0.18 96 Variant 31-27 8.1 VHH hShuffle 0.31 96 Variant 31-33 6 VHH hShuffle 0.32 96 Variant 30-40 7.1 VHH Shuffle 0.21 99 Variant 31-18 4.1 VHH hShuffle 0.36 99 Variant 30-5 9.3 VHH Shuffle 0.05 100

Example 15: VHH Libraries for GLP1R

A VHH library for GLP1R was developed similar to methods described in Example 14. Briefly, stable cell lines expressing GLP1R were generated, and target expression was confirmed by FACS. Cells expressing >80% of the target were then used for cell-based selections. Five rounds of cell-based selections were carried out against cells stably overexpressing the target of interest. 10⁸ cells were used for each round of selection. Before selection on target expressing cells, phage from each round was first depleted on 10⁸ CHO background cells. Stringency of selections was increased by increasing the number of washes in subsequent rounds of selections. The cells were then eluted from phage using trypsin, and the phage was amplified for the next round of panning. A total of 1000 clones from round 4 and round 5 are sequenced by NGS to identify unique clones for reformatting as VHH-Fc.

53 out of the 156 unique GLP1R VHH Fc binders had a target cell mean fluorescence intensity (MFI) value that was 2-fold over parental cells. The data for variant GLP1R-43-77 is seen in FIGS. 22A-22B and Tables 23B-24.

TABLE 23B Panning Summary VHH-Fc FACS binders Unique (MFI values 2-fold Library Phage over parental cells) VHH hShuffle 58  6 VHH Ratio/Shuffle 98 47

TABLE 24 GLP1R-43-77 Data Subset Name with Gating Path Count Median:RL1-A Sample E10.fcs/CHO-parent 11261  237 Sample E10.fcs/CHO-GLP1R 13684 23439

Example 16. GLP1R Libraries with Varied CDR's

A GLP1R library was created using a CDR randomization scheme.

Briefly, GLP1R libraries were designed based on GPCR antibody sequences. Over sixty different GPCR antibodies were analyzed and sequences from these GPCRs were modified using a CDR randomization scheme.

The heavy chain IGHV3-23 design is seen in FIG. 23A. As seen in FIG. 23A, IGHV3-23 CDRH3's had four distinctive lengths: 23 amino acids, 21 amino acids, 17 amino acids, and 12 amino acids, with each length having its residue diversity. The ratio for the four lengths were the following: 40% for the CDRH3 23 amino acids in length, 30% for the CDRH3 21 amino acids in length, 20% for the CDRH3 17 amino acids in length, and 10% for the CDRH3 12 amino acids in length. The CDRH3 diversity was determined to be 9.3×10⁸, and the full heavy chain IGHV3-23 diversity was 1.9×10¹³

The heavy chain IGHV1-69 design is seen in FIG. 23B. As seen in FIG. 23B, IGHV1-69 CDRH3's had four distinctive lengths: 20 amino acids, 16 amino acids, 15 amino acids, and 12 amino acids, with each length having its residue diversity. The ratio for the four lengths were the following: 40% for the CDRH3 20 amino acids in length, 30% for the CDRH3 16 amino acids in length, 20% for the CDRH3 15 amino acids in length, and 10% for the CDRH3 12 amino acids in length. The CDRH3 diversity was determined to be 9×10⁷, and the full heavy chain IGHV-69 diversity is 4.1×10¹².

The light chains IGKV 2-28 and IGLV 1-51 design is seen in FIG. 23C. Antibody light chain CDR sequences were analyzed for position-specific variation. Two light chain frameworks were selected with fixed CDR lengths. The theoretical diversities were determined to be 13800 and 5180 for kappa and light chains, respectively.

The final theoretical diversity was determined to be 4.7×10¹⁷ and the final, generated Fab library had a diversity of 6×10⁹. See FIG. 23D.

The purified GLP1R IgGs were assayed to determine cell-based affinity measurements and for functional analysis. FACS binding was measured using purified GLP1R IgG. As seen in FIG. 23E, the GLP1R IgG bound selectively to GLP1R-expressing cells with affinities in the low nanomolar range, demonstrating an IgG that selectively binds target expressing cell with an affinity of 1.1 nM. FACS binding was also measured in GLP1R IgGs generated using methods described in Examples 4-10. As seen in FIG. 23F, GLP1R IgGs bind selectively to GLP1R-expressing cells with affinities in the low nanomolar range.

cAMP assays using purified GLP1R IgG demonstrated that presence of GLP1R IgGs resulted in a left shift of the dose response curve of the GLP1 agonist induced cAMP response in GLP1R expressing CHO cells as seen in FIG. 23G. GLP1R IgGs generated using methods described in Examples 4-10 also resulted in a left shift of the dose response curve of the receptor agonist induced cAMP response in GLP1R expressing CHO cells (FIG. 23H).

The data shows the design and generation of GLP1R IgGs with improved potency and function.

Example 17. Oral Glucose Tolerance Mouse Model

The objective of this study was to evaluate the acute effects of a chimeric antibody GLP1R agonist and antagonist on glycemic control in a mouse model of diet induced obesity in C57BL/6J DIO mice. The test articles are seen below in Table 25.

TABLE 25 Test Article Identification GLP1 Agonist Ab GLP1 Antagonist Ab Ab Control Positive Control Identification GLP1R-59-2 GLP1R-3 GLP1R-2 Liraglutide Physical Clear Liquid Clear Liquid Clear Liquid Description Purity 95% 95% TBD Concentration 2.7 mg/ml 3.7 mg/ml TBD Storage Temperature Temperature Temperature Temperature Conditions set to maintain set to maintain set to maintain set to maintain 4° C. 4° C. 4° C. 4° C. Provided by Sponsor Sponsor Sponsor Testing Facility — = Not applicable.

For each test article, 7 different test article groups were generated as summarized in the following Table 26 with 8 animals per group.

TABLE 26 Experimental Design Dose Dose Test Dose Level Volume Concentration Dose Number of Group No. Material (mg/kg/day) (mL/kg) (mg/mL) Diet Regimen Route animals 1 GLP1R-2 0 5 0 HFD QD SC 8 2 Liraglutide 0.2 5 0.04 HFD QD SC 8 3 GLP1R-2 10 5 2 HFD QD SC 8 Liraglutide 0.2 5 0.04 4 GLP1R-59-2 10 5 2 HFD QD SC 8 5 GLP1R-59-2 10 5 2 HFD QD SC 8 Liraglutide 0.2 5 0.04 6 GLP1R-3 10 5 2 HFD QD SC 8 7 GLP1R-3 10 5 2 HFD QD SC 8 Liraglutide 0.2 5 0.04 No. = Number; ; HFD = high fat diet; QD = once daily; SC = Subcutaneous injection

On Day 3 (all animals) and Day 1 (Group 1-7), a non-fasting blood glucose was determined by tail snip. Approximately 5-10 μL of blood was collected. The second drop of blood from the animal was placed on a blood glucose test strip and analyzed using a hand-held glucometer (Abbott Alpha Trak).

After a non-fasting blood glucose measurement was made on the day of the procedure, the animals were weighed, tails marked, and the animals placed in clean cages without food. The animals were fasted for 4 hours and a fasting blood glucose measurement was determined. The animals were then treated with the indicated test article(s) as shown in Table 26.

The oral glucose tolerance test (OGTT) was administered to each animal 60 minutes later. The animals were dosed via oral gavage with 2 g/kg glucose (10 mL/kg). Blood glucose was determined via tail snip with the second drop of blood from the animal placed on a hand-held glucometer (Abbott Alpha Trak) at the following times relative to the glucose dose: 0 (just prior to glucose dose), 15, 30, 60, 90, and 120 minutes. Additional blood samples were obtained at the 15 minute and 60 minute time points of the OGTT for estimation of serum insulin.

FIGS. 24A-24B show GLP1R-3 inhibits GLP1:GLP1R signaling (FIG. 24A) with complete inhibition at higher concentrations (FIG. 24B). As seen in FIG. 24C, GLP1R-3 dosed animals maintained sustained high glucose levels after glucose administration, indicating GLP1:GLP1R signal blockade. As seen in FIG. 24D, GLP1R-59-2 dose at 10 mg/kg exhibited a sustained, low glucose levels similar to liraglutide control.

The data shows that the GLP1R antibodies generated have functional effects in a mouse model for glucose tolerance.

Example 18. GLP1R Agonists and Antagonists Effects in Wild-Type Mice

The effects of GLP1R-59-2 (agonist) and GLP1R-3 (antagonist) in wild-type mice were determined in this Example.

15 C57BL/6NHsd Mice were used and subjected to a Glucose Tolerance Test (GTT). The in vivo GTT test was performed on three groups of mice with 5 mice per group. All three groups were fasted for 13.5 hours before being weighed, time Zero Blood Glucose measured, and then injected i.p. with a 30% dextrose solution at a dose of 10 uL/gram body weight. Blood glucose measurements were recorded for each mouse at 15, 30, 60, 120, and 180 minutes after dextrose injection. A first group of mice were treated with GLP1R-59-2 at two doses: 10 mg/kg of GLP1R-59-2 at time of fasting (˜13.5 hrs. prior to GTT) and again two hours before start of GTT with 10 mg/kg of GLP1R-59-2. A second group of mice were treated with GLP1R-3 at two doses: 10 mg/kg of GLP1R-3 at time of fasting (˜13.5 hrs. prior to GTT) and again two hours before start of GTT with 10 mg/kg of GLP1R-3. A third group of mice were the control mice and were not treated. Data is seen for GLP1R-59-2 (agonist), GLP1R-3 (antagonist), and control in FIGS. 25A-25D. FIG. 25A shows the blood glucose levels in mice (y-axis) treated with GLP1R-59-2 (agonist), GLP1R-3 (antagonist), and control over time (in minutes, x-axis). FIG. 25B shows the blood glucose levels in mice (y-axis) treated with GLP1R-59-2 (agonist), GLP1R-3 (antagonist), and control. As seen in FIG. 25C, a significant reduction in blood glucose was observed in GLP1R-59-2 (agonist) treated mice in both the fasted (p=0.0008) and non-fasted (p<0.0001) mice compared to control. As seen in FIG. 25D, pre-dosed GLP1R-3 (antagonist) animals did not show decreased glucose in a 6 hour fast whereas control mice exhibited a decrease.

Example 19. Exemplary Sequences

Exemplary sequences of GLP1R are seen in Table 27. Table 27. GLP1R Sequences SEQ GLP1R Sequence ID NO: Variant

TABLE 27 GLP1R Sequences SEQ GLP1R ID NO: Variant Sequence 2411 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKEREFLAAITSGGATTYDD 01 NRKSRFTISADNSKNTAYLQMNSLKPEDTAVYYCWAALDGYGGRWGQGTLVTVSS 2412 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFRINRMGWFRQAPGKEREWVSTICSRGDTYYADS 02 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLDGYSGSWGQGTLVTVSS 2413 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRDFRVKNMGWFRQAPGKEREFVARITWNGGSAYY 03 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARILSRNWGQGTLVTVSS 2414 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMGWFRQAPGKEREFVAAISSGGRTSYADS 04 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYEGSWGQGTLVTVSS 2415 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYAMGWFRQAPGKEREFVAAISSGGRTRYADN 05 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYNGIWGQGTLVTVSS 2416 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGHTSDTYIMGWFRQAPGKEREFVSLINWSSGKTIYAD 06 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKGDYRGGYYYPQTSQWGQGTLVTVSS 2417 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKEREFVATIPSGGSTYYADS 07 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYNGSWGQGTLVTVSS 2418 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFGEFTMGWFRQAPGKERERVATITSGGSTNYADS 08 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVDDYSGSWGQGTLVTVSS 2419 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREVVAGIAWGDGITYYA 09 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNNWGQGTLVTVSS 2420 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSGVMGWFRQAPGKEREFVAAINRSGSTFYADS 10 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDWGQGTLVTVSS 2421 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGVTLDDYAMGWFRQAPGKEREFVAAINRSGSITYYA 11 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGSYDWGQGTLV TVSS 2422 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGLTFGIYAMGWFRQAPGKEREFVATISRSGASTYYAD 12 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYNDYDRGHDWGQGTLVTVSS 2423 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSDGMGWFRQAPGKERELVAAINRSGSTFYADS 13 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTARPGIFTTAPVEDWGQGTLVTVSS 2424 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTCGNYTMGWFRQAPGKERESVASITSGGRTNYADS 14 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLDGYTGSWGQGTLVTVSS 2425 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFNYYPMGWFRQAPGKEREWVATISRGGGTYYAD 15 NVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYSGIWGQGTLVTVSS 2426 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGIIGSFRTMGWFRQAPGKEREFVGFITGSGGTTYYADS 16 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAARRYGNLYNTNNYDWGQGTLVTVSS 2427 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAMGWFRQAPGKEREFVAAISWRGGSTNYAD 17 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATLGEPLVKYTWGQGTLVTVSS 2428 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGSFFSINAMGWFRQAPGKEREFVAGISSKGGSSTYYA 18 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSVGDWRWGQGTLVTV SS 2429 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGSRFSGRFNILNMGWFRQAPGKEREFVAAISRSGDTTY 19 YADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASLRNSGSNVEGRWGQGTLVTVS S 2430 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGGTSNSYRMGWFRQAPGKEREFVAVISWTGGSTYYA 20 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVALDGYSGSWGQGTLVTVSS 2431 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFNIGTYTMGWFRQAPGKEREFVAAIGSNGLANYAD 21 NVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYSGTWGQGTLVTVSS 2432 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSVYAMGWFRQAPGKEREFVAGIHSDGSTLYADS 22 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYMGTWGQGTLVTVSS 2433 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGNIKSIDVMGWFRQAPGKERELVAAVRWSGGITWYA 23 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVYYGDWEGSEPVQHEYDWGQGT LVTVSS 2434 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMGWFRQAPGKEREFVAAIYCSDGSTQYA 24 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAEALDGYWGQGTLVTVSS 2435 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGYTFRAYAMGWFRQAPGKEREMVAAMRWSGGITWY 25 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDYDGLPIKYDWGQGTLV TVSS 2436 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKERECVTAIFSDGGTYYADN 26 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYNGYWGQGTLVTVSS 2437 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGIHFAISTMGWFRQAPGKEREIVTAINWSGARTYYAD 27 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKFVNTDSTWSRSEMYTWGQGTLVTV SS 2438 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGLTFTSYAMGWFRQAPGKEREGVAVIDSDGTTYYAD 28 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYLDGYSGSWGQGTLVTVSS 2439 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSLPMGWFRQAPGKERELVAIRWSGGSTVYADS 29 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRWGQGTLVTVSS 2440 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSGVMGWFRQAPGKEREFVAAINRSGSTFYADS 30 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTWGQGTLVTVSS 2441 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWFRQAPGKERELVAAISSGGSTSYADS 31 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAMDGYSGSWGQGTLVTVSS 2442 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREYVAAISGSGSITNYAD 32 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANGIESYGWGNRHFNWGQGTLVTVSS 2443 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVAAIRWSGGITWYA 33 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAIFDVTDYERADWGQGTLVTVSS 2444 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFAFSGYAMGWFRQAPGKEREFVAAISWSGGITWYA 34 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAFVTTNSDYDLGRDWGQGTLVTVSS 2445 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGIPASIRTMGWFRQAPGKEREGVSWISSSDGSIYYADS 35 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVAALDGYSGSWGQGTLVTVSS 2446 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSLPMGWFRQAPGKERELVAIRWSGGSTVYADS 36 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRWDWGQGTLVTVSS 2447 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSWISTTDGSTYYA 37 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGIWGQGTLVTVSS 2448 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYAMGWFRQAPGKEREFVTAIDSESRTLYADS 38 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAALLDGYLGTWGQGTLVTVSS 2449 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMGWFRQAPGKEREFLGSILWSDDSTNYAD 39 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANLKQGSYGYRFNDWGQGTLVTVSS 2450 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGTIVNIHVMGWFRQAPGKERELVAAITSGGSTSYADN 40 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRHFEYDWGQGTLVTVSS 2451 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRSLGTYHMGWFRQAPGKEREGVSWISSSDGSTYYA 41 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVLDGYSGSWGQGTLVTVSS 2452 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDTGMGWFRQAPGKEREFVAAIRWSGKETWYA 42 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEDPSMYYTLEEYEYDWGQGTLVTV SS 2453 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMGWFRQAPGKERECVAAISSSDGRTYYAD 43 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGNWGQGTLVTVSS 2454 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMGWFRQAPGKEREFIATIFSGGDTDYADSV 44 KGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWDWGQGTLVTVSS 2455 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKEREIVASITSGGRKNYADS 45 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDDYSGSWGQGTLVTVSS 2456 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGHSFGNFPMGWFRQAPGKEREVIAAIDWSGGSTFYAD 46 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAKGIGVYGWGQGTLVTVSS 2457 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGSSFRFRAMGWFRQAPGKEREFVAAINRGGKISHYAD 47 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYIRPDTYLSRDYRKYDWGQGTLVTV SS 2458 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREGVAAIDSDGRTRYA 48 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTLVTVSS 2459 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGNILSLNTMGWFRQAPGKEREFVAGISWSGGSTYYAD 49 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLGNDWGQGTLVTVSS 2460 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGITFRRYDMGWFRQAPGKEREGVAYISSSDGSTYYAD 50 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDDYSGGWGQGTLVTVSS 2461 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGLTLSNYAMGWFRQAPGKEREFVAAISRSGSSTYYAD 51 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEMSGISGWDWGQGTLVTVSS 2462 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGYTTSINTMGWFRQAPGKEREVVAAISRTGGSTYYAD 52 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRRFEYDWGQGTLVTVSS 2463 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAMKPDGSITYYADS 53 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASASDYGLGLELFHDEYNWGQGTLVTV SS 2464 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGSIFSLNAMGWFRQAPGKERELVAGISSKGGSTYYAD 54 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWGQGTLVTVSS 2465 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYRMGWFRQAPGKEREAVAAIASMGGLTYYA 55 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYIGSWGQGTLVTVSS 2466 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTFGAFTMGWFRQAPGKERERVAAITCSGSTTYADS 56 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYNGSWGQGTLVTVSS 2467 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGIPSTIRAMGWFRQAPGKERESVGRIYWRDDNTYYAD 57 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSGSWGQGTLVTVSS 2468 GLP1R-40- EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREVVAGIAWGDGITYYA 58 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNNYYYPISRDEYDWGQGT LVTVSS 2469 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTIVPYTMGWFRQAPGKEREVVASISWSGKSTYYA 1 DSVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAQRRWSQDWGQGTQVTVSS 2470 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 2 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTGRGERDYWGQGTQVTVSS 2471 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTFSNYAMGWFRQAPGKEREFVATITWSGSSTYYA 3 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYREYGYWGQGTQVTVSS 2472 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFHINPMGWFRQAPGKEREfVAAINIFGTTNYADSV 4 KGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVDGGPLWDDGYDWGQGTQVTVSS 2473 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVASINIFGTTKYADSV 5 KGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAVGWGPHNDDRYDWGQGTQVTVSS 2474 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGTTFSIYAMEWFRQAPGKERELVATISRSGGTTYYAD 6 SVGGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAASWYYRDDYWGQGTQVTVSS 2475 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNFGTTKYADS 7 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAVRWGPHNDDRYDWGQGTQVTVSS 2476 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNFGTTKYADS 8 AKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPHNDDRYDWGQGTQVTVSS 2477 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFILYGYAMGWFRQAPGKEREGVSSISPSDASTYYAD 9 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLNTYSDSWGQGTQVTVSS 2478 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREGVTAISTSDGSTYYAD 10 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARDGYSGSWGQGTQVTVSS 2479 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGYTITNSYRMGWFRQAPGKEREFVAGITMSGFNTRY 11 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANRGLAGPAWGQGTQVTVSS 2480 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTFDDNAMGWFRQAPGKEREFVSGISTSGSTTYYAD 12 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAAGGYDYWGQGTQVTVSS 2481 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSYYHMGWFRQAPGKEREGVSWISSYYSSTYYA 13 DSESGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGYSCSWGQGTQVTVSS 2482 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSPFRLYTMGWFRQAPGKEREVVAHIYSYGSINYADS 14 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALWGHSGDWGQGTQVTVSS 2483 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFDTYGMGWFRQAPGKEREFVASITWSGSSTYYA 15 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANRIHWSGFYYWGQGTQVTVSS 2484 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTSSPYTMGWFRQAPGKEREFVSAISWSGGSTVYAD 16 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALIRRAPYSRLETWGQGTQVTVSS 2485 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFPINAMGWFRQAPGKEREGVAAITNFGTTKYADS 17 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDDHYDWGQGTQVTVSS 2486 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFDTYAMGWFRQAPGKEREFVAAITWGGGRTYY 18 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYRDYDYWGQGTQVTVSS 2487 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRRFSAYGMGWFRQAPGKEREFVAAVSWDGRNTYY 19 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASTDDYGVDWGQGTQVTVSS 2488 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFDNYAMGWFRQAPGKEREFVSAISGDGGTTYYA 20 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYRNRDYWGQGTQVTVSS 2489 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVSWITSFDASTYYAD 21 SVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTQVTVSS 2490 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVSTISTGGSSTYYAD 22 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTGRGRRDWGQGTQVTVSS 2491 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 23 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPVVPNTKDYWGQGTQVTVSS 2492 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGNVFMIKDMGWFRQAPGKEREWVTAISWNGGSTDY 24 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIVTYSDYDLGNDWGQGTQVTVSS 2493 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFPFSIWPMGWFRQAPGKEREFIATIFSGGDTDYADSV 25 KGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAYEEGVYRWDWGQGTQVTVSS 2494 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRGFSRYAMGWFRQAPGKEREFVAAIRWSGKETWY 26 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALGPVRRSRLEWGQGTQVTVSS 2495 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTSDIYGMGWFRQAPGKEREFVARIYWSSGNTYYA 27 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAYRFSDYSRPAGYDWGQGTQVTV SS 2496 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGNDFSFNSMGWFRQAPGKEREFLASVSWGFGSTYYA 28 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCARAYGNPTWGQGTQVTVSS 2497 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFTDYPMGWFRQAPGKERELESFVPINGTSTYYAD 29 SDSGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSCSWGQGTQVTVSS 2498 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVATISRGGSTTYYAD 30 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAGPRSGKDYWGQGTQVTVSS 2499 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFIFQLYVMGWFRQAPGKEREGVTYINNIDGSTYYAY 31 SVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRDGYSGSWGQGTQVTVSS 2500 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYAMEWFRQAPGKERELVATISRSGGRTYYAD 32 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAANWYYRYDYWGQGTQVTVSS 2501 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFPFRINAMGWFRQAPGKERELVTAISSSGSSTYYADS 33 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASGYYATYYGERDYWGQGTQVTVSS 2502 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTLSSYTMGWFRQAPGKEREFVSAISRGGGNTYYAD 34 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSYAEYDYWGQGTQVTVSS 2503 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYGMGWFRQAPGKEREGVAAINGGGDSTNYA 35 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAASASPYSGRNYWGQGTQVTVSS 2504 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGLtfSTTVMGWFRQAPGKEREGDGYISITDGSTYYADS 36 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAALDGYSGSWGQGTQVTVSS 2505 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTLENYRMGWFRQAPGKEREFVAAVSWSSGNAYY 37 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANWKMLLGVENDWGQGTQVTVS S 2506 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 38 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTVYGERDYWGQGTQVTVSS 2507 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSILSISPMGWFRQAPGKERELVAINFSWGTTDYADSv 39 KGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAYEQGVYRWDWGQGTQVTVSS 2508 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 40 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAERYRYSGYYARDSWGQGTQVTVS S 2509 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTLSDYAMGWFRQAPGKEREFVSAISRDGTTTYYA 41 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTSQYATDYWGQGTQVTVSS 2510 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRDLDYYVMGWFRQAPGKERELVAIKFSGGTTDYAD 42 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCADIAYEEGVYRWDWGQGTQVTVSS 2511 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFTFNAMGWFRQAPGKEREFVAGITRSAVSTSYAD 43 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAFRGIMRPDWGQGTQVTVSS 2512 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFDSYAMGWFRQAPGKEREFVAAITSSGGNTYYA 44 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPARYGARDYWGQGTQVTVSS 2513 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFNNDHMGWFRQAPGKEREFVAVIEIGGATNYAD 45 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATWDGRQVWGQGTQVTVSS 2514 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGTFRKLAMGWFRQAPGKERELVAAIRWSGGITWYA 46 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATLAKGGGRWGQGTQVTVSS 2515 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 47 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRAPSDRDYWGQGTQVTVSS 2516 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFRIYAMGWFRQAPGKERELVSSISWNSGSTYYAD 48 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAAYSYTQGTTYESWGQGTQVTVSS 2517 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFTSYRMGWFRQAPGKEREWMGTIDYSGRTYYA 49 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAMDGYSGSWGQGTQVTVSS 2518 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVAAINWNGDTTYYA 50 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRYSDYDYWGQGTQVTVSS 2519 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRFFSTRVMGWFRQAPGKERELVAIKFSGGTTDYADS 51 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAHEEGVYRWDWGQGTQVTVSS 2520 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 52 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSVYGTRDYWGQGTQVTVSS 2521 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSIDVMGWFRQAPGKEREGVSYISMSDGRTYYAD 53 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAELDGYSGSWGQGTQVTVSS 2522 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGLSFSGYTMGWFRQAPGKEREVVAAISRTGGSTYYA 54 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALIQRRAPYSRLETWGQGTQVTVSS 2523 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTLSIYGMGWFRQAPGKEREGVAAISWSDGSTSYAD 55 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVADIGLASDFDYWGQGTQVTVSS 2524 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSNYAMGWFRQAPGKEREFVATITRSSGNTYYAD 56 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPFKPYSYDYWGQGTQVTVSS 2525 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSIYTMGWFRQAPGKEREFVAAISGSSDSTYYADS 57 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATVPKTRYTRDYWGQGTQVTVSS 2526 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGNTFSSYAMGWFRQAPGKEREFVAIISRSGGRTYYAD 58 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAPYNETNSWGQGTQVTVSS 2527 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSTYAMGWFRQAPGKEREFVASISRSGGRTYYAD 59 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARYNERNSWGQGTQVTVSS 2528 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGTLNNNPMAMGWFRQAPGKEREFVVAIYWSNGKT 60 PYADSVKRRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSGAWGQGTQVTVSS 2529 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 61 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRAPSERDYWGQGTQVTVSS 2530 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFNNNDMGWFRQAPGKEREFVAVIKLGGATTYDD 62 YSEGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATWDARHVWGQGTQVTVSS 2531 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRAFSYYNMGWFRQAPGKEREGVSWISSSDGSTYYA 63 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGCSGSWGQGTQVTVSS 2532 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSTYAMGWFRQAPGKEREFVAAINRSGASTYYA 64 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALLGGRGGCGKGYWGQGTQVTVS S 2533 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSILDTYAMGWFRQAPGKERELVSGINTSGDTTYYAD 65 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLAGYEYWGQGTQVTVSS 2534 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTLSINAMGWFRQAPGKEREFVAHMSHDGTTNYAD 66 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCARLPNYRWGQGTQVTVSS 2535 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRLNAMGWFRQAPGKEREGVAAINNFDTTKYAD 67 SSKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRSDDRWGQGTQVTVSS 2536 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGLTNPPFDNFPMGWFRQAPGKEREFVAVISWTGGSTY 68 YAPSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCPAVYPRYYGDDDRPPVDWGQGTQ VTVSS 2537 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGPTFSKAVMGWFRQAPGKEREFVAAMNWSGRSTYY 69 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATPAGRGGYWGQGTQVTVSS 2538 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFSDYAMGWFRQAPGKEREFVATINWGGGRTYYA 70 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYARDYWGQGTQVTVSS 2539 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFILSDYAMGWFRQAPGKEREFVAAISSSEASTYYAD 71 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRFWAGYDSWGQGTQVTVSS 2540 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGYTDYKYDMGWFRQAPGKEREFVAAISWGGGLTVY 72 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVATVTDYTGTYSDGWGQGTQVT VSS 2541 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVATINWGGGNTYY 73 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYAYDYWGQGTQVTVSS 2542 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYYMGWFRQAPGKERELVAVILRGGSTNYAD 74 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARRYGNLYNTNNYDWGQGTQVTVS S 2543 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSILSSYVMGWFRQAPGKEREFVSAISRSGTSTYYADS 75 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYDRDYWGQGTQVTVSS 2544 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTLDNYAMGWFRQAPGKEREFVAAISWSGGSTYYA 76 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYSYDYWGQGTQVTVSS 2545 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGNTYSYKVMGWFRQAPGKEREFVGIIIRNGDTTYYAD 77 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASPKYMTAYERSYDWGQGTQVTVSS 2546 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRNYAMGWFRQAPGKEREFVATITTSGGNTYYAD 78 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYRRDWGQGTQVTVSS 2547 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTMGWFRQAPGKEREVVAAITGSGRSTYYA 79 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASAIGSGALRRFEYDWGQGTQVTVS S 2548 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGTFSAYAMGWFRQAPGKEREGVAAIRWDGGYTRY 80 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATTPTTSYLPRSERQYEWGQGTQV TVSS 2549 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 81 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSVYGERDYWGQGTQVTVSS 2550 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSFFSINAMGWFRQAPGKEREFVAGISQSGGSTAYAD 82 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAHRIVVGGTSVGDWRWGQGTQVTVS S 2551 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREMVASITSRKIPKYADS 83 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVWSGRDWGQGTQVTVSS 2552 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTFRRYVMGWFRQAPGKEREFVAAISRDGDRTYYA 84 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASTRLAGRWYRDSEYKWGQGTQVTV SS 2553 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFSDNAMGWFRQAPGKEREFVATISRGGSRTSYAD 85 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAGPRSGRDYWGQGTQVTVSS 2554 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFTFRSYAMGWFRQAPGKEREFVATITRNGDNTYYA 86 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATVGTRYNYWGQGTQVTVSS 2555 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSDYVMGWFRQAPGKERELISGITWNGDTTYYA 87 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVVRLGGYDYWGQGTQVTVSS 2556 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGIISNYHMGWFRQAPGKEREFVATITRSGGSTYYAD 88 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAMAGRGRWGQGTQVTVSS 2557 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGFSFDDDYVMGWFRQAPGKERELVSAIGWSGASTYY 89 ADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGSYDWGQGT QVTVSS 2558 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFPIYAMGWFRQAPGKEREWVSGISSRDDTTYYAD 90 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAHRIVFRGTSVGDWRWGQGTQVTVSS 2559 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRAFSYYNMGWFRQAPGKEREGVSWISSSDGSTYYA 91 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGYSGSWGQGTQVTVSS 2560 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSTFSIDVMGWFRQAPGKERELVAATGRRGGPTYYA 92 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAARTSYSGTYDYGVDWGQGTQVTVS S 2561 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAINWSGSITYYA 93 DSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVGRSGRDYWGQGTQVTVSS 2562 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNFGTTKYADS 94 VKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDDRYDWGQGTQVTVSS 2563 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGGTLNNNPMAMGWFRQAPGKEREFVVAIYWSNGKT 95 QYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTQVTVSS 2564 GLP1R-43- EVQLVESGGGLVQAGGSLRLSCAASGRTFNNDHMGWFRQAPGKEREFVAVIEIGGATNYAD 96 SVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASWDGRQVWGQGTQVTVSS 2565 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFAMGWMGWFRQAPGKEREFVARVSWDGRNAY 01 YANSRFGRFTISADNSKNTAYLQMNSLKPEDTAVYYCPRYVSPARDHGCWGQGTLVTVSS 2566 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGLTISTYIMGWFRQAPGKEREFVAVVNWNGDSTYYA 02 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGSYDWGQGTLV TVSS 2567 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWFRQAPGKERELVAAINRGGKITHYAD 03 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASLRNSGSNVEGRWGQGTLVTVSS 2568 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGVTLDLYAMGWFRQAPGKEREFVAAISPSAVTTYYA 04 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYDYYSDYPLPDANEYEWGQGTLVT VSS 2569 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYIMGWFRQAPGKEREFVAVINRSGSTTYYAD 05 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYSNSSDYYSQEGAYDWGQGTL VTVSS 2570 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYVMGWFRQAPGKEREGVSYISSSDGRTHYAD 06 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYNGSWGQGTLVTVSS 2571 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSRFGMGWFRQAPGKEREGVAAIGSDGSTSYADS 07 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASGRDRYARDLSEYEYVWGQGTLVTVSS 2572 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFRFNAMGWFRQAPGKEREFVAAINWRGSHPYYA 08 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATLGEPLVKYTWGQGTLVTVSS 2573 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGTFGVYHMGWFRQAPGKEREFLASVTWGFGSTYYA 09 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATTTRSYDDTYRNSWVYNWGQGTL VTVSS 2574 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFSFDDYAMGWFRQAPGKERELVAAIRWSGGITWYA 10 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSGSDYLPMDWGQGTLVTVSS 2575 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGPTFTIYAMGWFRQAPGKEREFVGAISMSGEDTIYADS 11 EKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYTSNTNYYNQEGAYDWGQGTLV TVSS 2576 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGPTFSNYYVGWFRQAPGKEREFVAAILCSGGITCYAD 12 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYIGTWGQGTLVTVSS 2577 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGTFSSIGMGWFRQAPGKEREGVAAIGSDGSTSYADS 13 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAASDRYARVLTEYEYVWGQGTLVTVSS 2578 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGVTFNNYGMGWFRQAPGKERELVAAIRWSGSATFYA 14 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADDGARGSWGQGTLVTVSS 2579 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFTMDGMGWFRQAPGKEREGVAAIGSDGSTSYAD 15 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGSNIGGSRWRYDWGQGTLVTVSS 2580 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGIFRFNAMGWFRQAPGKERELVAAISPAALTTYYAD 16 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYLPSPYYSSYYDSTKYEWGQGTLVT VSS 2581 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSGFSPNVMGWFRQAPGKEREVVAAISWNGGSTYYA 17 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRRFEYDWGQGTLVTVSS 2582 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFGFYAMGWFRQAPGKERELVAAISWSDASTYYA 18 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDYYNVSEYDWGQGTLV TVSS 2583 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYPMGWFRQAPGKERECVSTIWSRGDTYYADN 19 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSATWGQGTLVTVSS 2584 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAMGWFRQAPGKERELVAAISWSNDITYYA 20 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDYYSVSEYDWGQGTLVT VSS 2585 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYTMGWFRQAPGKEREFVAGIYNDGTASYYA 21 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFDGYTGNDWGQGTLVTVSS 2586 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGVTLDLYAMGWFRQAPGKEREWVARMYLDGDYPYY 22 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSGSWGQGTLVTVSS 2587 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTISRYIMGWFRQAPGKERELVAAINRSGKSTYYAD 23 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASTRFAGRWYRDSEYKWGQGTLVTVSS 2588 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLSVYAMGWFRQAPGKEREFVAAVRWSGGITWY 24 VDSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFDGYSGSDWGQGTLVTVSS 2589 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIFSITEMGWFRQAPGKERELVAAIAVGGGITWYADS 25 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHDVDDDESPYYSGGYYRALYDWGQG TLVTVSS 2590 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIYSLDAMGWFRQAPGKERELVAAISPAALTTYYAD 26 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASMSLRPLDPASYSPDIQPYDWGQGTL VTVSS 2591 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKERESVAAIDSDGRTHYAD 27 SVISRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGDWGQGTLVTVSS 2592 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLSfYAMGWFRQAPGKEREFVAAINRGGRISHYAD 28 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRRYGSPPHDGSSYEWGQGTLVTVS S 2593 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKEREFVAGISWTGGITYYA 29 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNVGFEWGQGTLVTVSS 2594 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMGWFRQAPGKEREGVAAIGSDGSTSYAD 30 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLRATITNFDEYVWGQGTLVTVSS 2595 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFNRYPMGWFRQAPGKEREFVAHMSHDGTTNYA 31 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAPGTRYYGSNQVNYNWGQGTLVTV SS 2596 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIFSFNAMGWFRQAPGKEREFVAGITRRGLSTSYADS 32 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAKGIGVYGWGQGTLVTVSS 2597 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGSISSINAMGWFRQAPGKERELVAGIITSGDSTYYAD 33 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGSAYVAGVRRRNAYHWGQGTLVTV SS 2598 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGTFSADVMGWFRQAPGKEREFVAAISTGSITIYADSV 34 KGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATYGYDSGLYFITDSNDYEWGQGTLVTVSS 2599 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDAAMGWFRQAPGKEREFVAAMRWRGGITWY 35 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGTLYDDYDGLPIKYDWGQGTLV TVSS 2600 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGDIFNINAMGWFRQAPGKEREPVAAISPAALTTYYAD 36 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATPIERLGLDAYEYDWGQGTLVTVSS 2601 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYNMGWFRQAPGKEREFVAAINWSGGITWYA 37 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPPDSSWYLDGSPEFFKWGQGTLV TVSS 2602 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSISVFDAMGWFRQAPGKERELVAGISGSGGDTYYAD 38 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASPKYSTHSIFDASPYNWGQGTLVTVS S 2603 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTSDDYAMGWFRQAPGKEREFVAALRWSSSNIDYT 39 YYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGHGDVSEYEYDWGQGTL VTVSS 2604 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSPNVMGWFRQAPGKEREFVAAITSSGETTWYAD 40 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDWGQGTLVTVSS 2605 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVAAINWSGDNTHY 41 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANWKMLLGVENDWGQGTLVTVSS 2606 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGDTFNCYAMGWFRQAPGKEREFVAVINWSGDNTHY 42 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGRYDWGQGT LVTVSS 2607 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSISTINVMGWFRQAPGKEREFVAAISPSAVTTYYADS 43 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGRGDVSEYEYDWGQGTLVTVSS 2608 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLSKYRMGWFRQAPGKEREFVAAIRWSGGITWYA 44 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIPHGIAGRITWGQGTLVTVSS 2609 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYAMGWFRQAPGKERELVAGIDQSGGITWYA 45 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADDYLGGDNWYLGPYDWGQGTLVT VSS 2610 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTIDDYAMGWFRQAPGKEREFVAAVSGTGTIAYYA 46 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYIDYDEALEETRGSYDWGQGTLV TVSS 2611 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFNNYVMGWFRQAPGKERELVAGITSGRDITYYA 47 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADGVLATTLNWDWGQGTLVTVSS 2612 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSGISFNAMGWFRQAPGKERELVAAISRSGDTTYYAD 48 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADLTTWADGPYRWGQGTLVTVSS 2613 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYAMGWFRQAPGKEREFVAAINRGGKISHYAD 49 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVRRYGNPPHDGSSYEWGQGTLVTVS S 2614 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYGMGWFRQAPGKERELVAIKFSGGTTDYADS 50 vkGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWGQGTLVTVSS 2615 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGGIFRFNAMGWFRQAPGKERELVAGISGSGGDTYYAD 51 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWGQGTLVTVSS 2616 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYAMGWFRQAPGKEREFVAAINRGGKISHYAD 52 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVRRYGSPPHDGSSYEWGQGTLVTVS S 2617 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSDFSLNAMGWFRQAPGKEREFVAAISWSGGSTLYA 53 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASNESDAYNWGQGTLVTVSS 2618 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLVNYDMGWFRQAPGKEREFVAAIRWSGGITWYA 54 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMLPPWGQGTLVTVSS 2619 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFEKDAMGWFRQAPGKEREMVAAIRWSGGITCYA 55 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSLPDDYDGLECEYDWGQGTLVT VSS 2620 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSFFKINAMGWFRQAPGKEREFVAGITRSGGSTYYAD 56 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAESLGRWWGQGTLVTVSS 2621 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAAIRWSGGITWYAD 57 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASHDSDWGQGTLVTVSS 2622 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAAIRWSGGITWYAD 58 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASHDSDYGGTNANLYDWGQGTLVTV SS 2623 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTDRSNVMGWFRQAPGKEREFVAAINRSGSTFYADS 59 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDWGQGTLVTVSS 2624 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSFFSINVMGWFRQAPGKERELVAATGRRGGPTYYA 60 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSVGDWRWGQGTLVTV SS 2625 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREGVAAIDSDGRTRYA 61 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGNWGQGTLVTVSS 2626 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGNIFSLNTMGWFRQAPGKEREFVAAINCSGNHPYYAD 62 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDDDGRDNWGQGTLVTVSS 2627 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWFRQAPGKEREFVAAVSGSGDDTYYAD 63 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYSSSSDYYSQEGAYDWGQGTLV TVSS 2628 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFPAYVMGWFRQAPGKERELLAVITRDGSTHYADS 64 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNGRWRIWSSRNPWGQGTLVTVSS 2629 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKERELVAVIGWGGKETW 65 YADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEDPSMGYYTLEEYEYDWGQGT LVTVSS 2630 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGPTFDTYVMGWFRQAPGKEREFVAAISMSGDDTAYA 66 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLRGRGDVSEYEYDWGQGTLVTVS S 2631 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVGAITWGGGNTYYA 67 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTDGDYDGWGQGTLVTVSS 2632 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGNTFSINVMGWFRQAPGKEREFVAAINWNGGSTDYA 68 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLDNDWGQGTLVTVSS 2633 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSTHWMGWFRQAPGKEREVVAVIYTSDGSTYYA 69 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANEYGLGSSIYAYKWGQGTLVTVSS 2634 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSISAMGWFRQAPGKEREFVAAISRSGGTTYYAD 70 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDEDYALGPNEYDWGQGTLVTVSS 2635 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSTFRINAMGWFRQAPGKERELVAAISPAALTTYYAD 71 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPYGSGSLYDDYDGLPIKYDWGQGT LVTVSS 2636 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVAAISWSNDITYYAD 72 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALSEVWRGSENLREGYDWGQGTLVT VSS 2637 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGLPVDYYAMGWFRQAPGKERELVAAISGSGDSTYYA 73 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQTEDSASIFGYGMDWGQGTLVTVS S 2638 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTLSTVNMGWFRQAPGKEREFVGAISRSGETTWYA 74 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVDCPDYYSDYECPLEWGQGTLVTVS S 2639 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFSFDDYAMGWFRQAPGKERELVAAVRWSGGITWY 75 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGDTGGAAYGWGQGTLVTVSS 2640 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSTLSINAMGWFRQAPGKEREGVSWISSSDGSTYYAD 76 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGRWGQGTLVTVSS 2641 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSSVSIDAMGWFRQAPGKEREFVAGISRSGDTTYYAD 77 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNNYYYPISRDEYDWGQGTL VTVSS 2642 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMGWFRQAPGKERELVAVTWSGGSTNYAD 78 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRWDWGQGTLVTVSS 2643 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYAMGWFRQAPGKEREFVAVVNWSGRRTYYA 79 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASSRMGVDDPETYGWGQGTLVTVSS 2644 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDAAMGWFRQAPGKEREFVAAVRWRGGITWY 80 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDYDGLPIKYDWGQGTLV TVSS 2645 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMGWFRQAPGKERELVASISRFGRTNYADSV 81 KGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANGIESWGQGTLVTVSS 2646 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREFVASITSGGRMWYA 82 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTLVTVSS 2647 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFRFSSYGMGWFRQAPGKEREGVAAIGSDGSTSYADS 83 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQVWGQGTLVTVSS 2648 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFDNYNMGWFRQAPGKEREFVAAISWNGVTIYYA 84 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDWGQGTLVTVSS 2649 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYSMGWFRQAPGKEREFVAAISSGGLKAYADS 85 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDDYSGSWGQGTLVTVSS 2650 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGYTFRAYVMGWFRQAPGKERELLAVITRDGSTHYAD 86 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNGRWRSWSSRNPWGQGTLVTVSS 2651 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVAAISRGSNSTDYAD 87 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYTDYDLWGQGTLVTVSS 2652 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTISSYAMGWFRQAPGKERELVAAISKSSISTYYADS 88 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALGPVRRSRLEWGQGTLVTVSS 2653 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGPTFDTYVMGWFRQAPGKEREFVAAISWTGDSSSDG 89 DTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAIFDVTDYERADWGQGTLV TVSS 2654 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFTLGNYAMGWFRQAPGKERELVSAITWSDGSSYYA 90 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASTRFAGRWGQGTLVTVSS 2655 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGNIDRLYAMGWFRQAPGKEREPVAAISPAAVTAGMT 91 YYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSGSYYYTDDELDWGQGTL VTVSS 2656 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFGRRAMGWFRQAPGKERELVAAIRWSGKETWY 92 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGNGGRTYGHSRARYEWGQGTLV TVSS 2657 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIGAMGWFRQAPGKEREYVGSITWRGGNTYYA 93 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGVTGGAAYGWGQGTLVTVSS 2658 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGLTFSTYWMGWFRQAPGKEREVVAVIYTSDGSTYYA 94 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATIDGSWREWGQGTLVTVSS 2659 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGFGIDfyAMGWFRQAPGKEREFVAAISGSGDDTYYAD 95 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASASDYGLGLELFHDEYNWGQGTLVT VSS 2660 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGNILSLNTMGWFRQAPGKEREFVASVTWGFGSTSYAD 96 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLGNDWGQGTLVTVSS 2661 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGSIYSLDAMGWFRQAPGKEREFVAAISPAALTTYYAD 97 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAGSSRIYIYSDSLSERSYDWGQGTLVTVS S 2662 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYGMGWFRQAPGKERELVAIKFSGGTTDYADS 98 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWDWGQGTLVTVSS 2663 GLP1R-41- EVQLVESGGGLVQPGGSLRLSCAASGRTFSKYAMGWFRQAPGKEREFVAAIRWSGGTTFYA 99 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGGWGTGRYNWGQGTLVTVSS 2664 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSIFSIYAMDWFRQAPGKEREFVAAISSDDSTTYYADS 01 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTAVLPAYDDWGQGTLVTVSS 2665 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSYISSSDGRTYYAD 02 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGLNGAAAAWGQGTLVTVSS 2666 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSNGPMGWFRQAPGKEREFVAHISTGGATNYADS 03 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQGWGQGTLVTVSS 2667 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRALSSYSMGWFRQAPGKEREFVALITRSGGTTFYAD 04 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRHSYVDWGQGTLVTVSS 2668 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSIGSINAMGWFRQAPGKEREFVAAISWSGGATNYAD 05 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASVAYSDYDLGNDWGQGTLVTVSS 2669 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGLSFDDYAMGWFRQAPGKEREFVAAISGRSGNTYYA 06 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALIQRRAPYSRLETWGQGTLVTVSS 2670 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMGWFRQAPGKEREGVAAISWSGGTTYYAD 07 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAAGWVAEYGYWGQGTLVTVSS 2671 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVATISSNGNTTYYAD 08 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADLRVLRLRRYEYNYWGQGTLVTVSS 2672 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFRSNAMGWFRQAPGKEREGVAAISTSGGITYYAD 09 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAERDGYGYWGQGTLVTVSS 2673 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKERELVAGISWNGGITYYA 10 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVRAGYDYWGQGTLVTVSS 2674 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREWVATISWSGGSTNYA 11 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVGRSGRDYWGQGTLVTVSS 2675 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRAFESYAMGWFRQAPGKEREFVAAIRWSGGSTYYA 12 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATGGWGTGRYNWGQGTLVTVSS 2676 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRIFSDYAMGWFRQAPGKEREFVATINGDGDSTNYAD 13 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANTYWYYTYDSWGQGTLVTVSS 2677 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRIFSDYAMGWFRQAPGKEREFVATINGDGDSTNYAD 14 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANTYCNYTYDSWGQGTLVTVSS 2678 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTLSRSNMGWFRQAPGKEREFVAAVRWSGGITWYA 15 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALGPVRRSRLEWGQGTLVTVSS 2679 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMGWFRQAPGKEREFVAAITWSGGSTNYA 16 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRAGRDSWGQGTLVTVSS 2680 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFNSYAMGWFRQAPGKEREFVAGITRSAVSTSYAD 17 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWGQGTLVTVSS 2681 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYVMGWFRQAPGKEREFVASITWSGGTTYYA 18 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRGSGRDYWGQGTLVTVSS 2682 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRALSSNSMGWFRQAPGKEREFVALITRSGGTTFYAD 19 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALNNRRRYVDWGQGTLVTVSS 2683 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYA 20 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVGRNGRDYWGQGTLVTVSS 2684 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISWSGGNTYYAD 21 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVPTIAYNTGYDYWGQGTLVTVSS 2685 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRIFDDYAMGWFRQAPGKERELVSGITWSGGSTYYA 22 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGYDGYDYWGQGTLVTVSS 2686 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKERELVSAISTDDGSTYYAD 23 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALPDDTYLATTYDYWGQGTLVTVSS 2687 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSIFSDNVMGWFRQAPGKEREMVAAIRWSGGITWYA 24 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGRGDVSEYEYDWGQGTLVTVS S 2688 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGEIASIIAMGWFRQAPGKEREWVSAINSGGDTYYADS 25 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRSRTIWPDWGQGTLVTVSS 2689 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSVSTMGWFRQAPGKEREIVAAITWSGSATYYAD 26 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQRRWSQDWGQGTLVTVSS 2690 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKERELVAGITGGGSSTYYAD 27 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTRYGYDYWGQGTLVTVSS 2691 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGIPFRSRTMGWFRQAPGKEREFVAGITRNSIRTRYADS 28 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAPRRPYLPIRIRDYIWGQGTLVTVSS 2692 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTIVPYTMGWFRQAPGKEREFVAAISWSGASTIYAD 29 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIGGTLYDRRRFEWGQGTLVTVSS 2693 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFSNNAMGWFRQAPGKEREGVAAINGSGSITYYAD 30 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAARDDYGYWGQGTLVTVSS 2694 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYGMGWFRQAPGKEREGVAGISWSDGSTSYAD 31 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAASDASFDYWGQGTLVTVSS 2695 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFSDYGMGWFRQAPGKEREGVASISWNDGSTSYA 32 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATADYDYWGQGTLVTVSS 2696 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSTYAMGWFRQAPGKERELVAAISWSSGTTYYAD 33 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLVTSDGVSEYNYWGQGTLVTVSS 2697 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFLFDSYAMGWFRQAPGKEREPVAAISPAALTTYYAD 34 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALEETRGSYDWGQGTLVT VSS 2698 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYAMGWFRQAPGKEREGVAAISWNSGSTYYA 35 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDARRYGYWGQGTLVTVSS 2699 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFGNYAMGWFRQAPGKEREFVAAISRSGSITYYAD 36 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDEDYALGPNEYDWGQGTLVTVSS 2700 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKERELVAGISWGGDSTYYA 37 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGNGYDYWGQGTLVTVSS 2701 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSYISSSDGRTYYAD 38 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSWGQGTLVTVSS 2702 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGLTFWTSGMGWFRQAPGKEREYVAAISRSGSLKGYA 39 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATVATALIWGQGTLVTVSS 2703 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFSINAMGWFRQAPGKERELVSGISWGGGSTYYAD 40 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVNEDGFDYWGQGTLVTVSS 2704 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFDDNAMGWFRQAPGKERELVAAISTSGSNTYYA 41 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAELREYGYWGQGTLVTVSS 2705 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFTSYNMGWFRQAPGKEREFLGSILWSDDSTNYAD 42 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQVWGQGTLVTVSS 2706 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYVMGWFRQAPGKEREFVAAINWNGSITYYA 43 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRSARNYWGQGTLVTVSS 2707 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISTSGGITYYAD 44 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDRIEYSRGGYDYWGQGTLVTVSS 2708 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFRKYAMGWFRQAPGKEREFVAAISSGGGSTNYA 45 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRYRERDSWGQGTLVTVSS 2709 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISWSGDTTYYAD 46 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIDLPDDTYLATEYDYWGQGTLVTVSS 2710 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSGFSPNVMGWFRQAPGKERELVAIKFSGGIIDYADS 47 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRWDWGQGTLVTVSS 2711 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTLTNHDMGWFRQAPGKEREGVSYISMSDGRTYYA 48 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSGSWGQGTLVTVSS 2712 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISRSGDSTYYAD 49 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTLDNYGYWGQGTLVTVSS 2713 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTASSYHMGWFRQAPGKEREFVAFIHRSGTSTYYAD 50 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADSITDRRSVAVAHTSYYWGQGTLVT VSS 2714 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGLTFSTYAMGWFRQAPGKEREIVAAITWSGGITYYAD 51 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHGSILLDRIEWGQGTLVTVSS 2715 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFSIYAMGWFRQAPGKERELVAAISSSGSITYYADS 52 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAAALDGPGDMYDYWGQGTLVTVSS 2716 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFDNYAMGWFRQAPGKERELVSGINSDGGSTYYA 53 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVPISSPSDRNYWGQGTLVTVSS 2717 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSLTAMGWFRQAPGKEREFVAAISPAALTTYYAD 54 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASRRAFRLSSDYEWGQGTLVTVSS 2718 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVAAVNWNGDSTYY 55 ADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANWKMLLGVENDWGQGTLVTVSS 2719 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMGWFRQAPGKERELVAGISSSGGSTYYAD 56 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGTYDYWGQGTLVTVSS 2720 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFDIYAMGWFRQAPGKERELVAAINRDDSSTYYAD 57 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGLGNYNYWGQGTLVTVSS 2721 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRSFSFNAMGWFRQAPGKERELVAAITKLGFRNYADS 58 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASIEGVSGRWGQGTLVTVSS 2722 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSFFSINAMGWFRQAPGKERELVSASTWNGGYTYYA 59 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSVGDWRWGQGTLVTV SS 2723 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYAMGWFRQAPGKEREFVAGITSSGGYTYYA 60 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVYYGDWEGSEPVQHEYDWGQGT LVTVSS 2724 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSIFSRNAMGWFRQAPGKEREFVAAIRWSGKETWYA 61 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDWGQGTLVTVS S 2725 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFDTYAMGWFRQAPGKEREFVAGISGDGTITYYAD 62 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDNPYWSGYNYWGQGTLVTVSS 2726 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTFSNYAMGWFRQAPGKERELVSGINSDGGSTYYA 63 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVSTNDGYDYWGQGTLVTVSS 2727 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGIYRVNTMGWFRQAPGKERELVAIKFSGGTTDYADS 64 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWDWGQGTLVTVSS 2728 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMGWFRQAPGKERELVAGISSSGSSTYYAD 65 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVSDGGYDYWGQGTLVTVSS 2729 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTSSIYNMGWFRQAPGKEREFVAAISRSGRSTSYADS 66 VKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLGNDWGQGTLVTVSS 2730 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRALSSYSMGWFRQAPGKEREFVALITRSGGTTFYAD 67 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDWGQGTLVTVSS 2731 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRALSRYGMVWFRQAPGKEREFVAAINRGGKISHYA 68 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGNGGRNYGHSRARYEWGQGTLVT VSS 2732 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGFKFNDSYMRWFRQAPGKEREFVVAINWSSGSTYYA 69 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVNGPIFWGQGTLVTVSS 2733 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTLSDYALGWFRQAPGKERELVSGINTSGDTTYYAD 70 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVTSSYDYWGQGTLVTVSS 2734 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFDIYGMGWFRQAPGKEREGVAAITGDGSSTSYAD 71 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADNDTEYGYWGQGTLVTVSS 2735 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGGTLDIYAMGWFRQAPGKEREFVAAISWSGSTTYYA 72 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGYDRDYWGQGTLVTVSS 2736 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRPYSYDAMGWFRQAPGKEREIVAAISRTGSSIYYAD 73 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDYDGLPIKYDWGQGTLVTV SS 2737 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGRTFRTYGMGWFRQAPGKEREGVAAISWSGNSTSYA 74 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARLSKRGNRSSRDYWGQGTLVTVSS 2738 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFDNYAMGWFRQAPGKERELVAGINWSDSSTYYA 75 DSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGWGEYDYWGQGTLVTVSS 2739 GLP1R-44- EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKERELVAGINWSDSSTYYAD 76 SVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTDYDEYNYWGQGTLVTVSS

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

What is claimed is:
 1. A nucleic acid library, comprising: a plurality of nucleic acids, wherein each of the nucleic acids encodes for a sequence that when translated encodes for a GLP1R binding immunoglobulin, wherein the GLP1R binding immunoglobulin comprises a variant of a GLP1R binding domain, wherein the GLP1R binding domain is a ligand for the GLP1R, and wherein the nucleic acid library comprises at least 10,000 variant immunoglobulin heavy chains and at least 10,000 variant immunoglobulin light chains, wherein the variant immunoglobulin heavy chain when translated comprises at least 90% sequence identity to SEQ ID NO: 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2317, 2318, 2319, 2320, or 2321, wherein the variant immunoglobulin light chain when translated comprises at least 90% sequence identity to SEQ ID NO: 2310, 2311, 2312, 2313, 2314, 2315, or
 2316. 2. The nucleic acid library of claim 1, wherein the nucleic acid library comprises at least 50,000 variant immunoglobulin heavy chains and at least 50,000 variant immunoglobulin light chains.
 3. The nucleic acid library of claim 1, wherein the nucleic acid library comprises at least 10⁵ non-identical nucleic acids.
 4. The nucleic acid library of claim 1, wherein the nucleic acid library comprises at least 100,000 variant immunoglobulin heavy chains and at least 100,000 variant immunoglobulin light chains. 